1. Cell Biology
Download icon

dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors

  1. Takunori Satoh
  2. Aya Ohba
  3. Ziguang Liu
  4. Tsuyoshi Inagaki
  5. Akiko K Satoh Is a corresponding author
  1. Hiroshima University, Japan
  2. Heilongjiang Academy of Agricultural Sciences, China
Research Article
Cited
12
Views
1,194
Comments
0
Cite as: eLife 2015;4:e06306 doi: 10.7554/eLife.06306

Abstract

In eukaryotes, most integral membrane proteins are synthesized, integrated into the membrane, and folded properly in the endoplasmic reticulum (ER). We screened the mutants affecting rhabdomeric expression of rhodopsin 1 (Rh1) in the Drosophila photoreceptors and found that dPob/EMC3, EMC1, and EMC8/9, Drosophila homologs of subunits of ER membrane protein complex (EMC), are essential for stabilization of immature Rh1 in an earlier step than that at which another Rh1-specific chaperone (NinaA) acts. dPob/EMC3 localizes to the ER and associates with EMC1 and calnexin. Moreover, EMC is required for the stable expression of other multi-pass transmembrane proteins such as minor rhodopsins Rh3 and Rh4, transient receptor potential, and Na+K+-ATPase, but not for a secreted protein or type I single-pass transmembrane proteins. Furthermore, we found that dPob/EMC3 deficiency induces rhabdomere degeneration in a light-independent manner. These results collectively indicate that EMC is a key factor in the biogenesis of multi-pass transmembrane proteins, including Rh1, and its loss causes retinal degeneration.

https://doi.org/10.7554/eLife.06306.001

eLife digest

The membranes that surround cells contain many proteins, and those that span the entire width of the membrane are known as transmembrane proteins. Rhodopsin is one such transmembrane protein that is found in the light-sensitive ‘photoreceptor’ cells of the eye, where it plays an essential role in vision.

Transmembrane proteins are made inside the cell and are inserted into the membrane surrounding a compartment called the endoplasmic reticulum. Here, they mature and ‘fold’ into their correct three-dimensional shape with help from chaperone proteins. Once correctly folded, the transmembrane proteins can be transported to the cell membrane. Incorrect folding of proteins can have severe consequences; if rhodopsin is incorrectly folded the photoreceptor cells can die, leading to blindness in humans and other animals.

Experiments carried out in zebrafish have shown that the chaperone protein Pob is required for the survival of photoreceptor cells. Pob is part of a group or ‘complex’ of chaperone proteins in the endoplasmic reticulum called the EMC complex. This suggests that the EMC complex may be involved in folding rhodopsin, but the details remain unclear.

Here, Satoh et al. studied the role of the EMC complex in the folding of rhodopsin in fruit flies. This involved examining hundreds of flies that carried a variety of genetic mutations and that also had low levels of rhodopsin. The experiments show that dPob—the fly version of Pob—and two other proteins in the EMC complex are required for newly-made rhodopsin to be stabilized. If photoreceptor cells are missing proteins from the complex, the light-sensitive structures in the eye degenerate.

Rhodopsin is known as a ‘multi-pass’ membrane protein because it crosses the membrane multiple times. Satoh et al. found that the EMC complex is also required for the folding of other multi-pass membrane proteins in photoreceptor cells. The next challenge will be to reveal how the EMC complex is able to specifically target this type of transmembrane protein.

https://doi.org/10.7554/eLife.06306.002

Introduction

In eukaryotes, most integral membrane proteins are synthesized, integrated into the membrane, and folded properly in the endoplasmic reticulum (ER). Molecular chaperones and folding enzymes are required for the folding of the integral membrane proteins in the ER. A comprehensive approach in yeast to identify genes required for protein folding in the ER identified the ER membrane protein complex (EMC), which comprises six subunits (Jonikas et al., 2009). Another report studying the comprehensive interaction map of ER-associated degradation (ERAD) machinery revealed that EMC contains four and three additional subunits in mammals and Drosophila, respectively (Christianson et al., 2011). The deletions of each emc1–6 gene causes the unfolded protein response (UPR), presumably caused by the accumulation of misfolded proteins (Jonikas et al., 2009). Meanwhile, a recent study showed that EMC also facilitates lipid transfer from ER to mitochondria (Lahiri et al., 2014).

In photoreceptors, the massive biosynthesis of rhodopsin demands chaperones in the ER. In the vertebrate retina, rhodopsin interacts with the ER degradation enhancing α-mannosidase-like 1 (EDEM1) protein and a DnaJ/Hsp40 chaperone (HSJ1B) (Chapple and Cheetham, 2003; Kosmaoglou et al., 2009). Meanwhile, in Drosophila photoreceptors, rhodopsin 1 (Rh1) sequentially interacts with chaperones calnexin99A (Cnx), NinaA, and Xport before exiting from the ER (Colley et al., 1991; Rosenbaum et al., 2006, 2011). Defects in rhodopsin biosynthesis and trafficking cause retinal degeneration in both Drosophila and humans; more than 120 mutations in the rhodopsin gene are associated with human retinitis pigmentosa (Mendes et al., 2005; Xiong and Bellen, 2013). The overwhelming majority of these mutations lead to misfolded rhodopsin, which aggregates in the secretory pathway (Hartong et al., 2006). Thus, it is important to understand the mechanisms underlying the folding and trafficking of rhodopsin as well as retinal degeneration caused by misfolded rhodopsin.

In zebrafish the partial optokinetic response b (pob)a1 mutant exhibits red cone photoreceptor degeneration (Brockerhoff et al., 1997; Taylor et al., 2005). The localization of overexpressed zebrafish Pob protein in cultured cells in the early secretory pathway including the ER and Golgi body indicates that Pob is involved in red cone rhodopsin transport (Taylor et al., 2005). The zebrafish pob gene is the homolog of a subunit of EMC, EMC3. Here we report the function of dPob, Drosophila pob homolog, on Rh1 maturation, photoreceptor maintenance, and expression of other multi-pass membrane proteins.

Results

dPob is essential for maturation and transport of Rh1

Retinal mosaic screening using the FLP/FRT method and two-color fluorescent live imaging was used to identify the genes essential for Rh1 maturation and transport (Satoh et al., 2013). For selected lines exhibiting defects in Rh1 accumulation in the live imaging screening, the immunocytochemical distribution of Rh1 was investigated to evaluate the phenotype with respect to transport and morphogenesis (Table 2, Satoh et al., 2013). Among them, CG6750e02662 (Kyoto stock number: 114504) exhibits severe Arrestin2::GFP and Rh1 reduction in rhabdomeres (Figure 1A,C) with normal ommatidial organization. CG6750e02662 has an insertion of a piggyBac transposon right downstream of the stop codon of CG6750 (Figure 1B). The phenotype was reverted by the precise excision of the piggyBac transposon or transgenically-expressed CG6750 (data not shown); this indicates Rh1 reduction is caused by reduced CG6750 gene function. CG6750 shares 65% identity and 82% similarity with zebrafish pob and 27% identity and 44% similarity with yeast EMC3. Because CG6750 is likely to be the homolog of zebrafish pob, we designated CG6750 as ‘dPob’ and analyzed its functions in Rh1 transport and retinal morphogenesis.

Identification of CG6750 as an essential gene for rhodopsin 1 (Rh1) biosynthesis.

(A) Observation of fluorescent protein localizations in CG6750e02662 mosaic retinas by the water immersion technique. RFP (red) indicates wild-type photoreceptors (R1–R8). Arrestin2::GFP (green) shows endogenous Rh1 localization in R1–R6 peripheral photoreceptors. (B) Schematic drawing of CG6750 and insertion/deletion mutants. The dPob-null mutant allele, dPob∆4, was created by the recombination of two FRTs on dPobf07762 and dPobCB−0279−3 using an FRT/FLP-based deletion method. (C, D) Immunostaining of dPobe02662 (C) and dPob∆4 (D) retinas expressing RFP as a wild-type cell marker (magenta) by anti-Rh1 antibody (green). Asterisks show mutant cells. Scale bar: 5 μm (A, C, D).

https://doi.org/10.7554/eLife.06306.003

To address the possibility that the severe reduction of Rh1 protein in dPobe02662 mutant is caused by the reduction of mRNA, Rh1 mRNA was quantified in whole-eye clones of the mutant. When compared with control FRT40A whole-eye clone, relative mRNA levels normalized to Act5C were, Rh1: 0.51 (n = 4, S.D. = 0.24); trp: 0.31 (n = 4, S.D. = 0.17); and Arr2: 0.49 (n = 4, S.D. = 0.24). Thus, the great reduction of the Rh1 protein level in dPobe02662 clones could not be interpreted by the reduction of mRNA.

As expected from the position of the insertion, dPob was still weakly expressed in dPobe02662 homozygous photoreceptors (Figure 2B,C), so it was classified as a hypomorphic allele. To further investigate the function of dPob, dPob∆4, a null mutant allele lacking the entire coding sequence of dPob, was created using an FRT/FLP-based deletion method (Figure 1B) (Parks et al., 2004). Unlike dPobe02662, which gives escapers up to the late pupal stage, dPob∆4 flies were lethal in the first instar larval stage. Immunostaining of dPob∆4 mosaic retinas shows a great reduction of Rh1 in dPob∆4 homozygous photoreceptors, similar to dPobe02662 homozygous photoreceptors (Figure 1D).

Construction of antisera against dPob.

(A) Immunoblotting of wild-type (+/+) and dPobe02662 homozygous (−/−) extracts from whole larvae using antiserum against dPob N- and C-terminal polypeptides. (B) Immunostaining of a dPobe02662 mosaic retina expressing RFP (red) as a wild-type cell marker (not shown) by rat anti-dPob-C1 antiserum (blue) and phalloidin (green). Asterisks show dPobe02662 homozygous photoreceptors. (C, D) Immunostaining of wild-type retinas by anti-dPob (green) and anti-NinaA (C) or anti-HDEL (D) antisera. Scale bar: 5 μm (BD).

https://doi.org/10.7554/eLife.06306.004

Next, antisera against dPob (Figure 2) were created to investigate dPob localization in fly photoreceptors. Four antisera (three against the N-terminal and one against the C-terminal) recognized a single ∼27 kD band in wild-type head homogenates by immunoblotting (Figure 2A). This band was greatly reduced in dPobe02662 homozygous head homogenates, indicating that these four antisera recognized dPob and that the molecular weight of dPob is ∼27 kD. In immunostaining dPobe02662 mosaic retinas, two of the C-terminal antisera (dPob-C1 and dPob-C3) produced similar staining patterns in the cytoplasm of wild-type cells which were reduced in dPobe02662 homozygous photoreceptors (Figure 2B and Figure 3B), indicating that these two antisera recognized dPob in tissue. Because dPob-C3 antiserum had the highest reactivity, we used it in further experiments. Anti-dPob reactivity co-localized with ER markers NinaA and HDEL (Figure 2C,D), indicating ER localization of dPob in fly photoreceptors.

dPob stabilizes rhodopsin 1 (Rh1) apoprotein.

(A) Immunostaining of a dPob∆4 mosaic retina from a fly reared in vitamin A (VA)-deficient medium by anti-Rh1 antibody. Asterisks show dPob∆4 homozygous photoreceptors. (BD) Immunostaining of a wild-type (B), ninaAp263(C), or dPob∆4 (D) ommatidium of flies reared in normal vitamin A-containing medium. (E) Immunostaining of a dPobe02662 mosaic retina in ninaAp263 homozygous mutant background from a fly reared in normal medium. Asterisks show dPob∆4 homozygous photoreceptors. Scale bar: 5 μm (AE).

https://doi.org/10.7554/eLife.06306.005

dPob is essential for the biosynthesis of Rh1 apoprotein

Rh1 comprises opsin (an apoprotein) and 11-cis retinal (a chromophore). Without the chromophore, newly synthesized Rh1 apoprotein accumulates in the ER as an N-glycosylated immature form (Ozaki et al., 1993). To investigate whether dPob is essential for the accumulation of Rh1 apoprotein in the ER, dPob∆4 mosaic retinas were observed in flies reared in medium lacking vitamin A, the source of the chromophore (Figure 3A). Rh1 apoprotein was greatly reduced in dPob∆4 photoreceptor cells, indicating that dPob is essential for the early stage of Rh1 biosynthesis before chromophore binding in the ER.

NinaA, the rhodopsin-specific peptidyl-prolyl-cis-trans-isomerase, is a known Rh1 chaperone. In contrast to dPob deficiency, which lacks both Rh1 apoprotein and mature Rh1 (Figure 3D), loss of NinaA causes accumulation of Rh1 apoprotein in the ER similar to that observed in the chromophore-depleted condition (Colley et al., 1991) (Figure 3C). To investigate the epistatic interaction between dPob and NinaA for Rh1 synthesis, Rh1 apoprotein was observed in the dPob∆4/ninaAp263 double mutant. Rh1 apoprotein was greatly reduced in dPob∆4/ninaAp263 double-mutant photoreceptors, similar to that in the dPob∆4 single mutant (Figure 3E). This indicates that dPob is epistatic to NinaA. Cnx is also an Rh1 chaperone and is known to be epistatic to NinaA. Rh1 apoprotein is greatly reduced in both the cnx1 mutant and cnx1/ninaAp269 double mutant (Rosenbaum et al., 2006), suggesting that dPob functions in the same stage or a stage close to that in which Cnx functions.

Other mutants with dPob-like phenotype

The null mutant of dPob shows a characteristic phenotype with no detectable protein expression of Rh1 and very weakened expression of other multiple-transmembrane domain proteins such as Na+K+-ATPase in the mosaic retina (see below). We did not find any other mutant lines with such a phenotype in the course of mosaic screening among 546 insertional mutants described previously (Satoh et al., 2013). To explore other mutants showing phenotypes similar to the dPob null mutant, we examined a collection of 233 mutant lines deficient in Rh1 accumulation in photoreceptor rhabdomeres obtained in an ongoing ethyl methanesulfonate (EMS) mutagenesis screening. The detail of the screening will be published elsewhere; at present the Rh1 accumulation mutant collection covers three chromosome arms, approximately 60% of the Drosophila melanogaster genome. Under the assumption of a Poisson distribution of the mutants on genes, the collection stochastically covers more than 80% of genes in those arms. The distribution of Rh1 and Na+K+-ATPase was examined for 55 lines of mutants on the right arm of the third chromosome, 93 lines of mutants on the right arm of the second chromosome, and 85 mutants on the left arm of the second chromosome. Among them, only two lines—665G on the right arm of the third chromosome and 008J on the right arm of the second chromosome—showed a dPob null-like phenotype in the mean distribution of Rh1 and Na+K+-ATPase in the mosaic retina (Figure 4A,C).

Loss of rhodopsin 1 (Rh1) apoprotein in EMC1 and EMC8/9 deficiency.

Immunostaining of a EMC1655G mosaic retina (A, B) or a EMC8/9008J mosaic retina (C, D) reared in normal (A, C) and vitamin A-deficient media (B, D). Asterisks show EMC1655G or EMC8/9008J homozygous photoreceptors. RFP (red) indicates wild-type photoreceptors (R1–R8). (A, C) Na+K+-ATPase, green; Rh1, blue; RFP, red. (B, D) Rh1, green; RFP, magenta. Scale bar: 5 μm (AD).

https://doi.org/10.7554/eLife.06306.006

Meiotic recombination mapping and RFLP analysis (Berger et al., 2001) were used to map the mutations responsible for the dPob-like phenotype of 008J and 655G. Close linkage of the mutation responsible for the dPob-like phenotype of 655G indicated that the responsible gene is located close to the proximal FRT. Since CG2943 gene, the potential Drosophila homolog of EMC1, is also close to the proximal FRT, CG2943 was recognized as a candidate of the responsible gene of 655G. As expected, Df(3R)BSC747, which is lacking the CG2943 gene, failed to complement the lethality of 655G. Targeted re-sequencing in the vicinity of CG2943 revealed that 655G has a two-base deletion at 3R:3729838-3729839 which causes a frame-shift mutation of CG2943, causing185aa deletion from I730 to C-terminus adding polypeptide of RTVRGQESGKQQCLEFLASSANAPRGAPVLYTAHNS. The only membrane-spanning helix of CG2943 is lost in this frame-shift mutation.

RFLP analysis narrowed down the cytology of the responsible gene of 008J to 58D2−59D11. Whole genome re-sequencing revealed that the 008J chromosome obtained three unique mutations in the mapped region compared with the starter stock: one silent mutation on CG30274 at 2R:18714026, a missense mutation on MED23 (E329K) at 2R:18777637, and one nonsense mutation on CG3501 at 2R:18770005 which turns Q40 to a stop codon. Complementation with the deficiencies over the MED23 (BSC783, BSC784) excluded the missense mutation on MED23 from the candidate mutation responsible for the dPob-like phenotype. The amino acid sequence of CG3501 shows 38% and 39% identity to the human EMC8 and EMC9, respectively, and no other gene similar to EMC8/9 was found in the Drosophila genome. Based on these results, we identified 655G and 008J as a loss of functional mutation of EMC1 and EMC8/9 of Drosophila and named these alleles EMC1655G and EMC8/9008J.

We investigated whether EMC1 and EMC8/9 are necessary for the accumulation of Rh1 apoprotein in the ER using EMC1655G and EMC8/9008J mosaic retinas reared in medium lacking vitamin A (Figure 4B,D). Rh1 apoprotein was greatly reduced in both EMC1655G and EMC8/9008J photoreceptor cells, indicating that EMC1 and EMC8/9 are also essential for the early stage of Rh1 biosynthesis, like dPob.

EMC1 binds to dPob and Cnx

To investigate if EMCs form a complex and bind to Rh1 apoprotein, we performed a co-immunoprecipitation assay (Figure 5). Since C-terminally tagged dPob protein did not predominantly localize to the ER in vivo (data not shown), GFP-tagged EMC1 protein (EMC1::GFP) was used as the bait. A protein-trap line expressing GFP-tagged sec61alpha protein (sec61::GFP) which localizes in the ER membrane was used as a negative control. Since the overall expression level of EMC1::GFP was strong, hs-Gal4 driver was used to activate UAS:EMC1::GFP for most of the experiments. To analyze the interaction between EMC1 and Rh1 apoprotein, Rh1-Gal4 driver was also used because the expression of EMC1::GFP was stronger in the photoreceptors (data not shown). For the Rh1-Gal4 experiment, flies were reared in a medium lacking vitamin A to accumulate Rh1 apoprotein in the ER. Membrane fraction was recovered from the adult heads, the membrane proteins extracted by CHAPS from the adult head membrane fraction were bound to anti-GFP magnetic beads, and the elutions were analyzed by immunoblotting with antibodies against GFP, Rh1, dPob, and Cnx.

Co-immunoprecipitation of EMC1::GFP with dPob and calnexin (Cnx).

Immunoblotting of precipitates with anti-GFP antibody from the head extract was prepared from Rh1-Gal4/UAS-EMC1::GFP or sec61::GFP flies reared in a vitamin A (VA)-deficient medium (left) or heat shock (hs)-Gal4/UAS-EMC1::GFP or sec61::GFP flies reared in a vitamin A-containing normal medium (right). The mature form of rhodopsin 1 (Rh1) is accumulated in the rhabdomeres in normal medium but not in vitamin A-deficient medium. Instead of the mature form, an N-glycosylated immature form of Rh1 with a larger molecular weight accumulated in the endoplasmic reticulum of flies reared in the vitamin A-deficient medium. In both input extracts prepared from Rh1-Gal4/UAS-EMC1::GFP or sec61::GFP flies there is a band with the same position as EMC1GFP; this band will be the protein cross-reacting to anti-GFP antibody.

https://doi.org/10.7554/eLife.06306.007

EMC1::GFP and sec61::GFP were concentrated in the immunoprecipitated extract from flies expressing either in the photoreceptor or in the whole head. dPob was co-immunoprecipitated with EMC1::GFP much more strongly than with sec61::GFP. Cnx was also well co-immunoprecipitated with EMC1::GFP but was barely detectable with sec61::GFP. However, Rh1 was not co-immunoprecipitated with EMC1::GFP from vitamin A-deficient photoreceptors accumulating immature Rh1 apoprotein in the ER. These results indicate that dPob and EMC1 are in a complex in vivo, as shown in yeast, and Cnx can also be associated with the complex, which is consistent with the result of epistatic analysis; the stage at which dPob works on the expression of Rh1 apoprotein is close to that of Cnx. Despite the requirement for the expression of Rh1 and co-localization with immature Rh1 apoprotein in the ER, EMC1 does not stably bind to Rh1, indicating that the EMC complex is only temporarily associated with Rh1 apoprotein.

EMC/dPob is required for the expression of multi-pass membrane proteins

To investigate the substrate specificity of EMC/dPob, we investigated the expressions of secreted or transmembrane proteins other than Rh1 in dPob∆4 mosaic retinas. In dPob∆4 photoreceptors, multi-pass membrane proteins, the alpha subunit of Na+K+-ATPase (Figure 6A) and transient receptor potential (TRP) (Figure 6B), were greatly reduced and neither anti-Rh3 nor anti-Rh4 staining was detected (Figure 6C,D). On the other hand, the type I single-pass membrane proteins Crb (Figure 6B) and DE-Cad (Figure 6E) were localized normally in the stalks and adherence junctions in dPob∆4 photoreceptors. Similarly, a type II single-pass membrane protein Nrt (Figure 6G) and a type VI single-pass membrane protein Syx1A (Figure 6H) were localized normally in Golgi units and on the plasma membrane in Pob∆4 photoreceptors. Eys, a secreted protein that expands the inter-rhabdomeric space (IRS) (Husain et al., 2006; Zelhof et al., 2006), was also secreted normally in dPob∆4 ommatidia, as expected from the near-normal size of the IRS (Figure 6I). Two other type I single-pass membrane proteins expressed in retinal cone cells, Neuroglian (Nrg) and Fasiclin III (FasIII), exhibited normal localization in contact sites between cone cells and cone cell feet (Figure 6J,K). Only one type II single-pass membrane protein, the beta subunit of Na+K+-ATPase (Nrv), showed deficient expression in Pob∆4 photoreceptors (Figure 6F). As alpha and beta subunits of Na+K+-ATPase are assembled into a heterodimer within the ER and then transported to the plasma membrane, the absence of Nrv in Pob∆4 photoreceptors can be interpreted as a consequence of the lack of the multi-pass alpha subunit. These results indicate that dPob is essential for the normal biosynthesis of multi-pass membrane proteins but not for single-pass membrane proteins or secreted proteins.

Essential role of dPob in the biosynthesis of multi-pass transmembrane proteins.

Immunostaining of a dPob∆4 mosaic retina (AH) or a dPobe02662 mosaic retina (I). Asterisks show dPob homozygous photoreceptors. (A) Na+K+-ATPase, green; Rh1, magenta. (B) Crb, green; TRP1, magenta. (C, D) Rh3 (C) and Rh4 (D), green; RFP (wild-type cell marker), magenta. Although the boundary between dPob∆4 and wild-type cells is unclear, all green signals are attached to RFP-expressing cell bodies, indicating that mutant R7 cells do not express Rh3 (C) or Rh4 (D). (E) DE-Cad staining. (F) Nrv, the beta subunit of Na+K+-ATPase, green; dMPPE, magenta. (G) Nrt staining. (H) Syx1A staining. (I) Eys staining. (J) Nrg, blue; F-actin, red; GFP-nls (wild-type cell marker), green. (K) FasIII staining. (L) Na+K+-ATPase, green; Rh1, magenta. Scale bar: 2 μm (A, B), 10 μm (C, D), 2 μm (EI).

https://doi.org/10.7554/eLife.06306.008

EMC1655G- and EMC8/9008J-deficient photoreceptors show similar substrate specificity to dPob∆4-deficient photoreceptors (Figure 6 and Figure 7). In both mutants, accumulation of the membrane proteins with multiple transmembrane domains, Na+K+-ATPase (Figure 4A,C), Rh3, Rh4 and TRP (Figure 7A,C), on the plasma membrane are greatly reduced in the photoreceptors. However, a type I single-pass transmembrane protein, Crb, is localized intensively in the stalks in EMC1655G or EMC8/9008J mutant photoreceptors (Figure 7B,D). A type II single-pass membrane protein, Nrt, and a type VI single-pass membrane protein, Syx1A, is localized normally in Golgi units and on the plasma membrane in EMC1655G and EMC8/9008J photoreceptors, respectively (Figure 7C,F). Eys was also secreted normally and formed a near-normal size of IRS in EMC1655G or EMC8/9008J mutant ommatidia (Figure 7B,D). Similar to Pob∆4 photoreceptors, a type II single-pass membrane protein, the beta subunit of Na+K+-ATPase (Nrv) was not detected in the plasma membrane of EMC1655G or EMC8/9008J photoreceptors (data not shown).

Essential role of EMC1 and EMC8/9 in the biosynthesis of multi-pass transmembrane proteins.

Immunostaining of a EMC1655G mosaic retina (A, B, C) or a EMC8/9008J mosaic retina (D, E, F). (A, D) Left: Rh3, middle: Rh4, right: TRP in green, RFP in magenda. (B, E) Eys in green, Crb in blue, and RFP, wild-type cell marker in red. (C, F) Left: dMPPE, middle: Nrt, right: Syx1A in green, RFP in magenda. Scale bar: 10 μm (left and middle in A, D), 5 μm (right in A, D), 5 μm (B, C, E, F).

https://doi.org/10.7554/eLife.06306.009

We observed the expression of dMPPE (Cao et al., 2011), a Golgi luminal metallophosphoesterase, anchored by a type II transmembrane helix in the N-terminal region and another transmembrane helix in the C-terminal region. dMPPE was expressed normally in Pob∆4, EMC1655G, and EMC8/9008J mutant photoreceptors (Figures 6F, 7C,F). As two transmembrane helices of dMPPE are separated from each other by the enzymatic domain, these two helices might not associate but behave as two separate transmembrane helices. The EMC requirement for proteins with two transmembrane helices therefore remains unclear.

ER membrane amplification in dPob-deficient photoreceptors

Electron microscopic observation of thin sections of late pupal flies showed massive amplification of the ER membrane in both dPobe02662 and dPob∆4 photoreceptors (Figure 8A–C) despite the substantial reduction in immature Rh1 apoprotein. In dPobe02662 photoreceptors the ER maintains its sheet structures: the number and length of the sheets was greatly increased but their lumens were almost normal with slight swelling and the sheets were aligned at a regular distance. Meanwhile, in dPob∆4 photoreceptors the ER sheet structures were no longer maintained and the cytoplasmic space was filled with ER membrane with a larger luminal space. Golgi bodies were also swollen and dilated, and sometimes vesiculated (Figure 8A–C, insets). Moreover, concordant with the reduction in Rh1, the rhabdomeres in dPob mutant photoreceptors were quite small and thin but the adherence junctions and basolateral membrane exhibited normal morphology. ER membrane amplification and rhabdomere membrane reduction therefore represent the most prominent phenotype in dPob-deficient photoreceptors.

Endoplasmic reticulum membrane amplification and unfolded protein response (UPR) induced in dPob∆4 photoreceptor.

(AC) Electron microscopy of late pupal photoreceptors: wild-type (A), dPobe02662 (B), and dPob∆4 photoreceptors (C). Arrow indicate adherens junctions. Insets show Golgi bodies. (D, E) Immunostaining of a dPobe02662 mosaic retina. dPob is shown in green and KDEL (D) or HDEL (E) are shown in magenta. Asterisks show dPob∆4 homozygous photoreceptors. Scale bar: 1 μm (AC), 5 μm (D, E).

https://doi.org/10.7554/eLife.06306.010

The massive amplification of the ER membrane in both dPobe02662 and dPob∆4 photoreceptors prompted us to quantify the amounts of residual ER proteins using anti-KDEL and HDEL antibodies. KDEL and HDEL sequences are signals for ER retention, and Drosophila ER resident chaperones including Hsp70–3 and PDI contain these sequences (Bobinnec et al., 2003; Ryoo et al., 2007). Corresponding to ER membrane amplification, anti-HDEL and anti-KDEL staining were greatly increased in dPob-deficient photoreceptors (Figure 8D,E).

Upregulated unfolded protein responses in dPob-deficient photoreceptors

Accumulation of unfolded proteins in the ER invokes the UPR, which includes activation of the transcription of chaperones and related genes, suppression of translation and enhanced degradation of unfolded protein. The UPR is regulated by some unique intracellular signal transduction pathways. Therefore, mutants lacking the function of a gene essential for folding or degradation of unfolded protein probably exhibit UPR. In fact, the yeast Pob homolog, EMC3, was identified by screening of mutants exhibiting upregulated UPR. ER amplification and chaperone induction, which we observed in dPob-deficient photoreceptors, are also common outcomes of UPR. We therefore examined whether UPR is induced in dPob-deficient photoreceptors. First we used the Xbp1:GFP sensor, which is an established method for detecting UPRs in flies (Ryoo et al., 2007). During UPR, Ire1 catalyzes an unconventional splicing of a small intron from the xbp1 mRNA, enabling translation into an active transcription factor (Yoshida et al., 2001). Using this mechanism, Xbp1:GFP sensor, a fused transcript of Drosophila Xbp1 and GFP translated only after the unconventional splicing by Ire1, can be used as a reporter of one of the UPR transduction pathways (Ryoo et al., 2007). In both dPob∆4 and dPobe02662 mutant mosaic retinas expressed Xbp1:GFP sensor in all R1−6 photoreceptors, and Xbp1:GFP fusion proteins were detected in the dPob mutant photoreceptors but not in the wild-type (Figure 9A and data not shown). Next, we examined the level of eukaryotic translation Initiation Factor 2α (eIF2α) phosphorylation because UPR is well known to induce eIF2α phosphorylation to attenuate protein translation on the ER membrane in a transduction pathway independent from IreI/Xbp1 (Ron and Walter, 2007; Cao and Kaufman, 2012). Anti-phospho-eIF2α signals were stronger in both dPob∆4 and dPobe02662 photoreceptors than in wild-type photoreceptors (Figure 9B and data not shown). These results indicate that UPR is induced in the dPob-deficient photoreceptors, similar to EMC mutant.

Unfolded protein response (UPR) induced in dPob∆4 photoreceptor.

(A) Projection image from the Z-series section with a 1 μm interval of dPob∆4 mosaic retina expressing RFP (magenta) as a wild-type cell marker and Xbp1:GFP as a UPR sensor. The Xbp1:GFP signal (green) is enhanced by immunostaining using anti-GFP antibody. Asterisks show dPob∆4 homozygous photoreceptors. (B) Immunostaining of a dPob∆4 mosaic retina expressing RFP (magenta) as a wild-type cell marker. Phosphorylated eukaryotic translation Initiation Factor 2α is shown in green. Asterisks show dPob∆4 homozygous photoreceptors.

https://doi.org/10.7554/eLife.06306.011

Rhabdomere development and degeneration in dPob null mutant

Because the synthesis of many membrane proteins was affected in dPob mutant cells, we observed the phenotype of dPob mutant throughout the developmental processes of photoreceptors. Despite the lack of many membrane proteins, ommatidial formation was not affected in dPob∆4 photoreceptors in mosaic retina; adherence junctions formed normally (Figure 6E) and the apical membrane was well differentiated into stalks and rhabdomeres (identified with Crb and phosphorylated moesin, respectively) (Figure 6B and data not shown) (Karagiosis and Ready, 2004). The IRS was formed normally and rhabdomeres were still separated by IRSs (Figure 8A–C). We observed dPob∆4 mosaic retinas at 58% and 75% pupal development (pd) by electron microscopy (Figure 10A,B). The wild-type photoreceptors at 58% pd had already begun to amplify the rhabdomere membranes. The rhabdomeres were shorter in dPob∆4 photoreceptors than in wild-type photoreceptors, but the difference in their appearance was subtle at this stage. Until 75% pd, the microvilli of wild-type rhabdomeres were ∼0.5 μm long and packed tightly. However, the microvilli of dPob∆4 rhabdomeres at 73% pd retained almost the same length and appearance as those at 58% pd, which is the same as the dPob∆4 rhabdomeres of the late pupal retina (Figures 10A,B and 8C). ER membrane expansion and dilation were already apparent at 58% pd. These results indicate that dPob does not inhibit overall photoreceptor development and morphogenesis but does affect microvilli elongation and rhabdomere formation.

Development and degeneration of dPob∆4 photoreceptor rhabdomeres.

Electron microscopy of pupal and adult dPob∆4 mosaic retinas. Asterisks show dPob∆4 homozygous photoreceptors. Scale bar: 1 μm. (A, B) dPob∆4 mosaic ommatidia from 58% pupal development (A) and 73% pupal development (B) under constant light (L) condition. (CF) dPob∆4 mosaic ommatidia from flies reared in complete darkness (D) (C, E) or under 12 hr light/12 hr dark conditions (D, F). Ommatidia from 3-day-old (C, D) and 17-day-old (E, F) flies. (D, inset) dPob∆4 R5 photoreceptor rhabdomere at higher magnification.

https://doi.org/10.7554/eLife.06306.012

Because zebrafish pob was identified as the responsible gene of poba1 mutant which exhibits red cone photoreceptor degeneration (Brockerhoff et al., 1997; Taylor et al., 2005), we investigated photoreceptor degeneration of the dPob null mutant. Three-day-old dPob∆4 mosaic retinas from flies reared under dark or 12 hr light/12 hr dark cycles were observed by electron microscopy (Figure 10C,D). In both conditions the rhabdomeres of dPob∆4 photoreceptors invaginated into the cytoplasm, indicating that dPob-deficient rhabdomeres undergo retinal degeneration in a light-independent manner, like Rh1 null mutants (Kumar and Ready, 1995). No microvilli or invaginations were observed in 17-day-old dPob∆4 mosaic retinas, suggesting most invaginated microvilli had degraded before day 17 (Figure 10E,F). Such rhabdomere degeneration was observed not only in R1–6 peripheral photoreceptors but also in R7 central photoreceptors. Therefore, dPob is an essential protein for maintenance of retinal structure, similar to the zebrafish pob gene.

Discussion

The present study shows that dPob, the Drosophila homolog of a subunit of EMC, EMC3, localizes in the ER and is essential for Rh1 accumulation of the rhabdomeres. The deficiency of each of two other EMC subunits, EMC1 and EMC8/9, also shows absence of Rh1 on the rhabdomeres. Mammalian EMC8 and EMC9 were identified together with EMC7 and EMC10 by high-content proteomics strategy (Christianson et al., 2011). Unlike EMC1−6 subunits, EMC8 and EMC9 do not have a transmembrane helix or signal peptide and no experimental data have been reported to show the functions of these subunits. We observed that Drosophila EMC8/9-deficient cells lack accumulation of Rh1 apoprotein in the ER and impaired biosynthesis of the multi-pass transmembrane proteins. These phenotypes in EMC8/9 deficiency are indistinguishable from those in dPob and EMC1 mutant cells, suggesting that EMC8/9 work together with EMC1 and dPob. This is the first functional study of the additional subunits of EMC, which are lacking in yeast.

We found that null mutants of EMC subunits are defective in expressing the multi-pass transmembrane proteins rhodopsins, TRP, and the alpha subunit of Na+K+-ATPase, which have seven, six, and eight transmembrane helices, respectively. In contrast, the EMC null mutants adequately express type I, type II, or type IV single-pass membrane proteins. Our observation on the substrate specificity of EMC is mostly consistent with previous reports. Jonikas et al. (2009) found that EMC mutants and a strain overexpressing a misfolded transmembrane protein, sec61-2p or KWS, had a similar genetic interaction pattern and suggested that EMC works as a chaperone for transmembrane proteins. A recent study in Caenorhabditis elegans using a hypomorphic EMC6 allele and RNAi knock-down of emc1–6 genes showed results partially consistent with our study; at least two pentameric Cys-loop receptors, AcR and GABAA, consisting of subunits with four transmembrane helices, were significantly decreased in the hypomorphic EMC6 mutants but GLR-1, a tetrameric AMPA-like glutamate receptor with four transmembrane helices and a type I single-pass transmembrane EGF receptor, was not affected (Richard et al., 2013). Despite its four transmembrane helices, GLR-1 was normally expressed in the hypomorphic emc6 mutant of the nematode; however, these results may indicate that the residual activity of EMC was sufficient for the expression of GLR-1. The degree of requirement of EMC activity can vary for each membrane protein. In fact, in a dPob hypomorphic allele, dPobe02662, near-normal expression of Na+K+-ATPase was detected (Figure 6I) despite a severe reduction in a dPob null allele, dPob∆4. Overall, the results observed in the dPob null mutant does not conflict with previous studies but rather clarifies the role of EMC in the biosynthesis of multi-pass transmembrane proteins. Because of the limited availability of antibodies, we could not show a clear threshold for the number of transmembrane helices in the substrates for EMC activity. In total, the data presented to date indicate that EMC affects the expression of membrane proteins with four or more transmembrane helices.

Co-immunoprecipitation of dPob/EMC3 and Cnx by EMC1 indicates that EMC components and Cnx can form a complex. The photoreceptors of an amorphic mutant of Cnx show complete loss of Rh1 apoprotein (Rosenbaum et al., 2006), just as shown in dPob, EMC1 or EMC8/9 mutants. Moreover, both Cnx and EMC3 are epistatic to the mutant of the rhodopsin-specific chaperone, NinaA, which accumulates Rh1 apoprotein in the ER. These results indicate that EMC and Cnx can work together in the Rh1 biosynthetic cascade prior to NinaA. Cnx, the most studied chaperone of N-glycosylated membrane proteins, recognizes improperly folded proteins and facilitates folding and quality control of glycoproteins through the calnexin cycle, which prevents ER export of misfolded proteins (Williams, 2006). One possible explanation for our result is that the EMC-Cnx complex is required for multi-pass membrane proteins to be incorporated into the calnexin cycle. If the EMC-Cnx complex is a chaperone of Rh1, physical interaction is expected between ER-accumulated Rh1 apoprotein and the EMC-Cnx complex. Indeed, it is reported that Cnx is co-immunoprecipitated with Drosophila Rh1 (Rosenbaum et al., 2006). However, in this study, Rh1 apoprotein accumulated in the chromophore-depleted photoreceptor cells was not co-immunoprecipitated with EMC1. Thus, even if EMC is a Rh1 chaperone, our result indicates that EMC is unlikely to be working in the calnexin cycle or acting as a buffer of properly folded Rh1 apoprotein ready to bind the chromophore 11-cis retinal.

In addition to preventing the export of immature protein by the calnexin cycle, Cnx is also known to recognize the nascent polypeptides co-translationally (Chen et al., 1995). The dual role of Cnx might explain the observations that both dPob/EMC3 and Cnx are epistatic to another ER resident chaperone, NinaA, whereas Cnx but not the EMC-Cnx complex binds to Rh1. These results imply that the EMC-Cnx complex is more likely to be involved in the earlier processes such as membrane integration or co-translational folding than in the folding of fully translated membrane-integrated Rh1 apoprotein.

In spite of the absence of Rh1 apoprotein, UPR is much more upregulated in the EMC3 null mutant than in the NinaA null mutant which accumulates Rh1 apoprotein in the ER. The elevated UPR without accumulation of Rh1 apoprotein in the dPob mutant photoreceptor can be explained either by the quick degradation of Rh1 apoprotein or by accumulation of the single-pass membrane proteins abandoned by the multi-pass binding partner.

Newly synthesized secreted proteins co-translationally translocate across the membrane through the translocons Sec61 in eukaryotic ER or SecYEG in the plasma membrane of bacteria. The translocons also mediate integration of the transmembrane helix of the integral membrane protein into the lipid bilayer (Park and Rapoport, 2012). In bacteria, mitochondria and chloroplasts, YidC/Oxa1/Alb3 proteins specifically facilitate insertion, folding, and assembly of many transmembrane proteins (Wang and Dalbey, 2011). In the ER membrane of eukaryotes, in addition to the translocon, other components such as translocon-associated protein/signal sequence receptor (TRAP/SSR) complex and translocating chain-associating membrane protein (TRAM) complex are required for the membrane insertion of the transmembrane helix. Most of the newly synthesized multi-pass membrane proteins are co-translationally integrated into the ER membrane through the translocon complex. Although the mechanism of this process is yet to be fully understood, it is assumed that only one or two transmembrane helices can be stored in the translocon channel and the lateral gate and that the next set of newly synthesized transmembrane helices displace them (Rapoport et al., 2004; Cymer et al., 2014). In the case of nascent chain of bovine rhodopsin, translocon associates with transmembrane helices sequentially, and TRAM temporarily associates with the second transmembrane helix (Ismail et al., 2008). EMC may be involved in these co-translational membrane integration or co-translational folding processes.

Zebrafish pob was identified as the responsible gene of poba1 mutant, which exhibits red cone photoreceptor degeneration (Brockerhoff et al., 1997; Taylor et al., 2005). Because only red cone photoreceptors degenerated in zebrafish poba1 mutant, pob is postulated as a gene with a red cone-specific function. However, the identification of the poba1 mutation as hypomorphic together with pob expression in all photoreceptors, as well as its localization in the early secretory pathway, suggests that Pob has a general function rather than being red cone-specific (Taylor et al., 2005). We found that dPob-deficient rhabdomeres undergo retinal degeneration in a light-independent manner, like Rh1 null mutants (Kumar and Ready, 1995). Rhabdomere degeneration was observed not only in R1–6 peripheral photoreceptors but also in R7 central photoreceptors. Our results indicate that dPob is an essential protein for the maintenance of retinal structure, similar to the zebrafish pob gene.

Materials and methods

Drosophila stocks and genetics

Flies were reared at 20–25°C in 12 hr light/12 hr dark cycles and fed standard cornmeal/glucose/agar/yeast food unless noted otherwise. Vitamin A-deficient food contained 1% agar, 10% dry yeast, 10% sucrose, 0.02% cholesterol, 0.5% propionate, and 0.05% methyl 4-hydroxybenzoate.

UAS-Xbp1::GFP was a gift from H Ryoo at New York University and other Drosophila stocks obtained from Bloomington Stock Center (BL) or the Kyoto Drosophila Genetic Resource Center (KY) are referred to with their respective sources and stock numbers.

dPob deletion mutants were made using a standard induced FLP/FRT recombination method (Parks et al., 2004). Trans-heterozygous PBac(WH)f07762 (BL19109) and P (RS3)CB−0279−3 (KY123106) males carrying hs-FLP (BL6876) were heat treated three times at 37°C for 1 hr at larval stages. SM6a-balanced offspring were genotyped using PCR to select the recombinant carrying both the proximal side of PBac(WH)f07762 and the distal side of P (RS3)CB−0279−3 with the following primers: 5′-CTCCTTGCCAGCTTCTGC-3′ and 5′-TCGCTGTCTCACTCAGACTCA-3′ for P (RS3)CB−0279−3, and 5′–CCACCGAAGAGGCCTACTATT-3′ and 5′-TCCAAGCGGCGACTGAGATG-3′ for PBac(WH)f07762.

Transgenic flies for UAS-dPob, UAS-EMC1::GFP

The entire coding region of the dPob gene was amplified from a cDNA clone LD37839 (DGRC: Drosophila Genomics Resource Center, Bloomington, IN, USA) and cloned into pTW (DGRC) to construct pP{UAST-dPob}. To construct pP{UAST-EMC1::GFP}, the entire coding region of CG2943 except the stop codon was amplified from a cDNA clone LD19064 (DGRC) and cloned into pTWG (DGRC). Plasmids were injected into embryos by BestGene Inc. (Chino Hills, CA, USA) to generate transgenic lines.

Live imaging of fluorescent proteins expressed in photoreceptors

Fluorescent proteins expressed in photoreceptors were imaged by water-immersion technique.

y w ey-FLP;CG6750e02662 FRT40A/ CyO y+ (KY114504) was mated with w;P3RFP FRT40A/SM1;Rh1-Arrestin2::GFP eye-FLP/TM6B (Satoh et al., 2013). Late pupae of the siblings with GFP-positive RFP mosaic retina were attached to the slide glass using double-sided sticky tape and the pupal cases around the heads were removed. The pupae were chilled on ice, embedded in 0.5% agarose, and observed using an FV1000 confocal microscope equipped with a LUMPlanFI water-immersion 40× objective (Olympus, Tokyo, Japan). Arrestin2::GFP specifically binds to activated rhodopsin (Satoh et al., 2010). Rh1 was activated by a 477 nm solid-state laser to bind Arr2:GFP and GFP. The wild-type marker P3RFP is DsRed gene under the control of three Pax3 binding sites and labels photoreceptors (Bischof et al., 2007).

EMS mutagenesis and screening

The precise method of screening, whole genome re-sequencing, will be described elsewhere. Briefly, second or third chromosomes carrying P-element vector with FRT on 40A, 42D, or 82B (Berger et al., 2001) were isogenized and used as the starter strains. EMS was fed to males in a basic protocol (Bökel, 2008) and mosaic retinas were generated on F1 or F2. The estimated number of lethal mutations introduced per chromosome arm was 0.8–1.8. The mutants were screened based on the distribution of Arr2-GFP by confocal live imaging under water-immersion lens using 3xP3-RFP as the wild-type marker, as previously described for the screening of insertional mutants (Satoh et al., 2013).

Mapping and determination of mutations

Meiotic recombination mapping was carried out by the standard method (Bökel, 2008). Briefly, to allow meiotic recombination between the proximal FRT, the phenotype-responsible mutation and a distal miniature w+ marker, flies carrying isogenized chromosome of 008J and 655G were crossed with flies with isogenized P{EP755} and P{EP381} which carry miniature-w+ marker, respectively. Female offspring carrying the mutated chromosome and the miniature-w+-marked chromosome were crossed with males carrying FRT42D, P3RFP, and Rh1Arr2GFP. The resulting adult offspring with w+ mosaic, which means maternally inherited both FRT and w+, were observed using live imaging to judge whether the mutation responsible for the dPob-like phenotype had been inherited. The recovered flies were individually digested in 50 µl of 200 ng/µl Proteinase K in 10 mM Tris-Cl (pH 8.2), 1 mM EDTA, and 25 mM NaCl at 55°C for 1 hr and heat inactivated at 85°C for 30 min and at 95°C for 5 min. 0.5 µl of the digested solution were used as the template of PCR amplification for RFLP analysis according to the method described in the FlySNP database (Chen et al., 2008; http://flysnp.imp.ac.at/index.php). The mutation responsible for the dPob-like phenotype of 008J was mapped between SNP markers 1417 and 1518 defined in the FlySNP database.

Whole-genome and targeted re-sequence of EMS-generated mutants

For the whole genome re-sequencing of the 008J mutant, the second chromosome was balanced over a balancer, CyO, P{Dfd-GMR-nvYFP}(Bloomington stock number 23230) to facilitate the isolation of homozygous embryo. Using REPLI-G single cell kit (QIAGEN, Hilden, Germany), the genomic DNA was amplified from two 008J homozygous embryos independently. A sequencing library was prepared using Nextera DNA sample preparation kit (Illumina, San Diego, CA, USA) for each embryo and 2 × 250 bp reads were obtained using MiSeq v2 kit (Illumina). Reads were mapped to release five of the Drosophila melanogaster genome using BWA 0.7.5a. The RFLP-mapped region of 008J was covered by reads with an average depth of 23.2× and width of 99.5%. Mapped reads were processed using picard-tools 1.99 and Genome Analysis Tool Kit 2.7-2 (GATK, Broad Institute, Cambridge, MA, USA). SNVs and Indels were called using Haplotypecaller in GATK. SNVs and Indels were subtracted by the ones of the isogenized starter stock to extract the unique variants in 008J and annotated using SnpSift (Cingolani, 2012). The point mutation on 2R:18770005 was verified by capillary sequencing of PCR-amplified fragment using 5′ GTCGCGGTCACACTTTCTAG 3′ and 5′ CTGCAGCGTCATCAGTTTGT 3′ as primers.

For targeted re-sequencing of 655G, a region including CG2943 was amplified from a heterozygous fly of the 655G mutant chromosome and the starter chromosome using KOD FX Neo DNA polymerase and 5′ TTTTGTTCTTGTTGGGCGACTCCTTTTCCGTCTC 3′ and 5′ AGGCTGTGTCTTTGTTGTTTTGGCGTTGTCGTC 3′ as primers. Reads covering the CG2943 gene region at a depth of 2213–6436 were obtained using MiSeq and mapped, as described above. The sequence was confirmed by capillary sequencing and PCR using 5′ GCAAGAATCCCATCGAGCAT 3′ and 5′ CCTTCTTCACGTCCCTGAGT 3′ as primers.

Antisera against dPob and CNX99a

Fragments of cDNA encoding V28-D104 (dPob-N) or G173-S247 (dPob-C1) of dPob were amplified from a cDNA clone, LD37839 (Drosophila Genomics Resource Center, Bloomington, IN, USA) and cloned into pDONR-211 using Gateway BP Clonase II and then into pET-161 expression vector using Gateway LR Clonase II (Life Technologies, Carlsbad, CA, USA). The fusion proteins with 6xHis-tag were expressed in BL21-Star (DE3) (Life Technologies) and purified using Ni-NTA Agarose (QIAGEN). To obtain antisera, rabbits were immunized six times with 300 µg dPob-N fusion protein (Operon, Tokyo, Japan) and three rats were immunized six times with 125 µg dPob-C1 fusion protein (Biogate, Gifu, Japan). Antisera against Drosophila Cnx were raised by immunizing a rabbit four times with 400 to 200 µg of synthetic peptide corresponding to C-terminal 24 amino acids of Cnx99a protein conjugated to KLH (Sigma Aldrich Japan, Tokyo, Japan).

Immunoblotting

Immunoblotting was performed as described previously (Satoh et al., 1997). The antibodies used were as follows: rabbit anti-dPob–N-terminal (dPob-N) (1:2000 concentrated supernatant) (made by the authors of this paper), three rat anti-dPob–C-terminal antibodies (dPob-C1-3) (1:2000 concentrated supernatant) (made by the authors of this paper) as primary antibodies. HRP-conjugated anti-rat or anti-rabbit IgG antibody (1:20,000, Life Technologies) was used as a secondary antibody. For co-immunoprecipitation, 1:2000 rabbit anti-dPob-N, 1:2000 rabbit anti-Cnx99A, 1:2000 rabbit anti-GFP (Life Technologies), mouse anti-Rh1 monoclonal antibody 4C5, and detected by biotinylated secondary antibodies followed by HRP-conjugated avidin. Signals were visualized using enhanced chemiluminescence (Clality Western blotting ECL Substrate; BioRad, Hercules, CA, USA) and imaged using ChemiDoc XRS+ (BioRad).

Immunohistochemistry

Fixation and staining were performed as described previously (Satoh and Ready, 2005). The primary antisera were as follows: rabbit anti-Rh1 (1:1000) (Satoh et al., 2005), chicken anti-Rh1 (1:1000) (Satoh et al., 2013), mouse monoclonal anti-HDEL (1:100) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-KDEL (1:100) (Assay Designs, Ann Arbor, MI, USA), rabbit anti-NinaA (1:300) (gift from Dr Zuker, Colombia University), mouse monoclonal anti-Na+K+-ATPase α subunit (1:500 ascite) (DSHB, Iowa City, IA, USA), rat monoclonal anti-DE-cad (1:20 supernatant) (DSHB), mouse monoclonal anti-Syx1A (1:20 supernatant) (DSHB), mouse monoclonal anti-Nrt (1:20 supernatant) (DSHB), mouse monoclonal anti-Nrv (1:20 supernatant) (DSHB), mouse monoclonal anti-FasIII (1:20 supernatant) (DSHB), mouse monoclonal anti-Nrg (1:20 supernatant) (DSHB), mouse monoclonal anti-Chp (24B10) (1:20 supernatant) (DSHB), rat anti-Crb (gift from Dr Tepass, University of Toronto), rabbit anti-TRP (gift from Dr Montell, Johns Hopkins University), rabbit anti-dMPPE (1:50) (gift from Dr Han, Southeast University), and rabbit anti-phosphorylated eIF2α (1:300) (Cell Signaling Technologies, Danvers, MA, USA). The secondary antibodies used were anti-mouse, rabbit, rat, and chicken IgG labeled with Alexa Fluor 488, 568, and 647 (1:300) (Life Technologies) and Cy2 (1: 300) (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Samples were examined and images recorded using a FV1000 confocal microscope (60×, 1.42-NA lens; Olympus, Tokyo, Japan). To minimize bleed-through, each signal in double- or triple-stained samples was imaged sequentially. Images were processed in accordance with the guidelines for proper digital image handling using ImageJ and/or Adobe Photoshop CS3.

Co-immunoprecipitation analysis of EMC complex

The EMC1 gene was cloned into a P-element vector pTWG using the Gateway System (Life Technologies) to express EMC1 protein-tagged GFP on the C-terminus under control of upstream activation sequence (UAS). Transgenic lines were generated by the BestGene Inc. (Chino Hills, CA, USA). UAST-EMC1-GFP(1M), a line carrying the transgene on the second chromosome, was crossed to Rh1-Gal4 line to express EMC1-GFP in the photoreceptor or to hs-Gal4 line to express EMC1-GFP in the whole body. A protein-trap line, Sec61alpha [ZCL0488] which constitutively expresses GFP-tagged Sec61alpha protein, was used as a control. To accumulate rhodopsin in the ER, flies were reared in the vitamin A-deficient medium in a Rh1-driven experiment. For heat-shock driven expression, newly eclosed adult fly flies were incubated at 37°C for 45 min a day before preparation. Within 0–1 days after eclosion, flies were frozen with liquid nitrogen and stored at −80°C. The heads were collected by sieving in liquid nitrogen, ground to powder and homogenized in buffer (50 mM Tris-Cl, 500 mM NaCl, pH 7.5) containing 1:200 Protein inhibitor cocktail VI (Calbiochem, San Diego, CA, UAS) using BioMasher II (Wako Pure Chemical, Osaka, Japan) with motor drive. Debris was removed by centrifugation at 950×g for 5 min and the membrane was precipitated by centrifugation at 21,500×g for 15 min. Approximately 30 µl of membrane pellet were solubilized by 130 µl of 1% CHAPS and placed on ice for 1 hr, and the insoluble membrane was removed by centrifugation at 21,500×g for 30 min. The extract was diluted fivefold by the buffer and 50 µl of Anti-GFP-Magnetic beads (MBL, Nagoya, Japan) were added and mixed by mild rotation for 18 hr. The magnetic beads were rinsed with 2× 100 μl of 0.1% CHAPS in buffer and the bound protein was extracted by incubation in 20 µl SDS-PAGE Sampling Buffer (BioRad) for 5 min at room temperature and an equal amount of Sampling Buffer with 2-mercaptoethanol was then added. The extracts were heat denatured for 5 min at 37°C. SDS-PAGE and immunoblotting was performed as described above.

Electron microscopy

Electron microscopy was performed as described previously (Satoh et al., 1997). Samples were observed on a JEM1200 or JEM1400 electron microscope (JEOL, Tokyo, Japan).

Quantification of relative expression of mRNA of Rh1, TRP, and Arr2 normalized by Act5C

Whole-eye mutant clones were generated using the FRT/GMR-hid method (Stowers and Schwarz, 1999). Both eyes were dissected from two adult flies per sample and cDNA was reverse-transcribed using SuperPrep Cell Lysis and RT Kit for qPCR (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Eyes with whole-eye clones of FRT40A were used as a control to obtain the relative standard curves. qPCR reactions were performed using the StepOne real-time PCR system (Life Technologies) and KOD SYBR qPCR Mix (Toyobo, Osaka, Japan), according to the manufacturers’ instructions. PCR condition was 98°C for 2 min, followed by 40 cycles at 98°C for 15 s, 55°C for 15 s, and 68°C for 45 s, and a melt curve stage of 95°C for 30 s, 60°C for 1 min, and 0.3°C/s increments to 98°C, with primers of Rh1: (ninaE-qF1:5′-GTGGACACCATACCTGGTC-3′ and ninaE-qR1:5′-GCGATATTTCGGATGGCTG-3′), Arr2: (Arr2-qF1:5′-AAGGATCGCCATGGTATCG-3′ and Arr2-qR1:5′-TACGAGATGACAATACCACAGG-3′), TRP: (Trp-qF2:5′-GAATACACGGAGATGCGTC-3′ and Trp-qF2:5′-CTCGAGTTCCATGGATGTG-3′), Act5C: (5′-GCTTGTCTGGGCAAGAGGAT-3′ and 5′-CTGGAACCACACAACATGCG-3′). The relative expression levels were normalized by Act5C.

References

  1. 1
  2. 2
  3. 3
  4. 4
    A new form of inherited red-blindness identified in zebrafish
    1. SE Brockerhoff
    2. JB Hurley
    3. GA Niemi
    4. JE Dowling
    (1997)
    The Journal of Neuroscience 17:4236–4242.
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
    Mechanisms of integral membrane protein insertion and folding
    1. F Cymer
    2. G von Heijne
    3. SH White
    (2014)
    Journal of Molecular Biology, 10.1016/j.jmb.2014.09.014.
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
    Rhodopsin plays an essential structural role in Drosophila photoreceptor development
    1. JP Kumar
    2. DF Ready
    (1995)
    Development 121:4359–4370.
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
    In situ inhibition of vesicle transport and protein processing in the dominant negative Rab1 mutant of Drosophila
    1. A Satoh
    2. F Tokunaga
    3. S Kawamura
    4. K Ozaki
    (1997)
    Journal of Cell Science 110:2943–2953.
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
    A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype
    1. RS Stowers
    2. TL Schwarz
    (1999)
    Genetics 152:1631–1639.
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44

Decision letter

  1. David Ron
    Reviewing Editor; University of Cambridge, United Kingdom

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Congratulations: we are very pleased to inform you that your article, “dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors”, has been accepted for publication in eLife, subject to revisions of grammar and text. We also hope you would give due consideration to the minor comments of reviewers 1 and 3. The Reviewing Editor for your submission was David Ron.

Reviewer #1

In Figure 5, right panel it is not clear why the sec61::GFP input shows a band the size of the EMC1::GFP; what is the nature of this band?

Equally why is the +/- vitamin A experiment performed with different driver lines? wouldn't it be better to do both with the Rh1-Gal4 driver? Please give the rationale of using the two drivers.

Reviewer #3

1) The authors show that EMC1::GFP does not co-immuno-precipitate with Rh1. Does calnexin co-IP with Rh1? I ask because the authors conclude (in the Discussion) that “ EMC is unlikely to be working in the calnexin cycle”, and it is difficult for me to understand why the authors have come to that conclusion.

2) The authors report negative results with the ERAD component mutants, EDEM1, EDEM2 and VCP/+. I don't see the point of showing such negative data, as one cannot draw any conclusions. Perhaps the individual EDEM mutants do not show Rh1 phenotype as they are redundant (in mammals, EDEMs are known to be genetically redundant). Also, VCP's effect was assessed in a heterozygous condition, and a negative result here simply means that there is no dominant genetic effect. How about analyzing homozygous clones? Alternatively, since ERAD is not a major point of this work, the authors may want to take out this part, as it does not contribute to the overall story.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The previous decision letter after peer review is shown below.]

Thank you for choosing to send your work entitled “dPob is essential for rhodopsin maturation in Drosophila photoreceptors” for consideration at eLife. Your full submission has been evaluated by Randy Schekman (Senior editor) and 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors. Between them, the three reviewers had expertise in Drosophila genetics, the study of assembly and maturation of membrane transporters and ER protein folding homeostasis. Their decision was reached after discussions between the reviewers.

The reviewers noted the significance of your discovery of a role for a Drosophila member of the EMC complex in the maturation of rhodopsin. Your discovery that dPob/EMC3 acts upstream of ninaA in rhodopsin maturation was specifically credited with supporting a role for this member of the EMC complex in the early steps of the maturation of a multi-pass membrane protein. However, the expert reviewers were unanimous in their view that these finding do not add up to an advance of sufficient measure to merit publication in eLife.

It is possible that a different manuscript, for example one with further details on genetic lesions in other Drosophila EMC homologs or one that carried the biochemical analysis of the dPob/EMC3 lesion further would had received a warmer welcome at eLife. Unfortunately, the manuscript before us is too far off this mark to suggest specific experiments that would render it suitable for this journal.

Thus we are left with no choice but to return the manuscript to you in the hope that you will find the reviewers’ comments (appended below) of use.

Reviewer #1

A mosaic screen for mutations in (otherwise essential) genes required for rhodopsin trafficking and maturation in the fly eye led the authors to dPod, in whom homozygosity for a hypomorphic allele compromised Rh1 expression in the rhabdomeres.

dPod is a fly homolog of a zebra fish gene mutations in which compromise color vision and of EMC3 a yeast gene whose compromise activates the unfolded protein response and whose encoded protein forms a stable complex with several other proteins that collectively constitute the ER Membrane Complex (or EMC).

Since its identification in 2009, the EMC has been postulated to play a role in the biogenesis of transmembrane proteins. This paper provides several pieces of information that support that notion.

The most important findings pertain to:

1) The failure of Rh1 to accumulate in the ER of dPod mutant flies that are deprived of vitamin A.

2) The epistatic relationship between dPod and ninaA, wherein the lack of dPod preempts the accumulation of Rh1 normally obsereved in ninaA mutants.

3) The selectivity of dPod mutations in compromising the trafficking of multi-pass transmembrane proteins, whilst preserving that of secreted and single pass proteins.

Together these findings point to site of dPod action early during the biogenesis of multi-pass transmembrane proteins.

Other findings, such as the altered ER morphology in mutant tissue and the activation of the UPR are less informative and more anticipated by the yeast work.

Thus the crucial issue for the reviewers is to establish the strength of the experimental data and, importantly the degree to which it advances our understanding of the EMC's biological role. If the application of this Drosophila genetic system has provided strong evidence favoring a selective role for this EMC component in early biogenesis of multi-pass transmembrane proteins, then the paper would be of interest to a broad readership regardless of the degree to which these findings were anticipated by prior speculation. If, however, the additional experimental data derived from the Drosophila system were deemed merely incremental, the paper would be more suited for a specialist journal.

Reviewer #2

In this manuscript, Satoh and colleagues report a genetic analysis of dPob, a Drosophila homolog of zebrafish Pob and yeast EMC4. The data presented here supports the idea that this gene is involved in the maturation of rhodopsins and other multipass transmembrane domain proteins in the endoplasmic reticulum. Consistently, the loss of dPob leads to the activation of the Unfolded Protein Response, and a defect in rhabdomere development.

EMC genes have been identified through a large-scale yeast genetic interaction screen, but their precise physiological roles had remained unclear. A couple of reports, based on studies in zebrafish and C. elegans, indicate that they are involved in membrane protein maturation. Although the authors have done a solid job in their characterization of dPob function in Drosophila, it appears to be an extension of the C. elegans work (Richard et al., 2013), but with more analysis of potential substrates. Moreover, this study does not address many of the pressing questions regarding the EMC complex. Does dPob's role in Drosophila reflect the entire EMC complex function? What is the mechanistic basis of the specificity of dPob towards multispan membrane proteins? Based on these grounds, whether the overall novelty and scope of this study justifies the publication of eLife can be subject to debate. Below are a few specific comments along these lines.

1) The authors had identified dPob through a previously reported genetic screen (Satoh et al., 2013). Were there other EMC homologs identified in the screen? If it is the case, the authors should highlight those results, as it would indicate that the observed effect is not an isolated function of dPob.

2) EMC genes are grouped because of their genetic interactions with each other in yeast. This study is potentially significant because the function of EMCs still remain poorly understood. Naturally, one must ask whether other EMC gene homologs show similar effects in rhodopsin maturation or not. The authors can examine this through in vivo RNAi, or better, by use of available mutant alleles.

3) Does dPob physically interact with rhodopsin and other multipass membrane proteins? Immuno-precipitation experiments may be useful.

4) Is there any mechanistic insight as to how dPob would specifically recognize multipass membrane proteins? Although the authors show the dPob locus in Figure 1, they do not describe any domain structures in the gene. The fact that dPob specifically affects transmembrane domain is an intriguing phenomenon, but a mechanistic insight is lacking.

5) Does dPob physically interact with rhodopsins and other targets? A simple immune-precipitation experiment should provide answers.

Reviewer #3

The manuscript by Satoh et al. adds dPob, a subunit of the Drosophila EMC complex, to the list of factors required for rhodopsin biogenesis, an important process with biomedical relevance. The authors show that in the absence of dPob several rhodopsins do not mature and their steady-state levels are strongly decreased. Besides rhodopsins, two other polytopic membrane proteins, Na, K-ATPase alpha subunit and the TRP channel, were affected while four proteins with a single transmembrane segment and one secreted protein were not affected. These are interesting observations that do indeed suggest a role of dPob in the early biogenesis of polytopic membrane proteins.

However, I have major doubts whether the set of experiments adds much to our understanding of the specific function of dPob. Chaperone activity and protein folding are mentioned all over the manuscript but neither are ever addressed directly. Instead, steady-state levels of proteins and induction of the UPR are used as proxies to infer the folding status of the putative substrates. Both phenomena could be rather indirectly caused by lack of dPob function. In my opinion the manuscript does not achieve more than narrowing down which proteins are affected by loss of dPob function in a non-systematic fashion. The numbers of substrates in the affected and unaffected classes are low (furthermore, 'with a single membrane-spanning domain' is not an accurate topological classification for a membrane protein). Potentially, the distinctions between affected and unaffected proteins could fall into completely different categories: half-life of the precursor, activity of the precursor, trafficking machinery used by the precursor to leave the ER, activity of the precursor at the ER to name just a few speculative ideas. Therefore, the last sentences of the Conclusion section are strongly over-stated.

This work is solid and interesting. It presents a good set of figures. I don't perceive the manuscript as outstanding because the actual function of dPob is inferred from correlations with highly complex steady-state effects on selected individual proteins or on global ER homeostasis. From an outstanding paper I would expect either a systematic and unbiased approach to identifying the substrates of the putative chaperone complex or a delineation of the features that it recognizes in its substrates or some direct evidence for the actual chaperone activity proposed.

https://doi.org/10.7554/eLife.06306.013

Author response

We made some changes regarding the reviewers’ comments.

Reviewer #1

In Figure 5, right panel it is not clear why the sec61::GFP input shows a band the size of the EMC1::GFP; what is the nature of this band?

We think this band indicated a protein cross-reacting to anti-GFP antibody. We added the following sentence to the figure legend.

In both input extracts prepared from Rh1-Gal4/UAS-EMC1::GFP or sec61::GFP flies, there is the band with the same position to EMC1::GFP: this band will be the protein cross-reacting to anti GFP antibody

Equally why is the +/- vitamin A experiment performed with different driver lines? wouldn't it be better to do both with the Rh1-Gal4 driver? Please give the rationale of using the two drivers.

Since the overall expression level of EMC1::GFP was strong, hs-Gal4 driver was used to activate UAS:EMC1::GFP for the most of experiment. To analyze the interaction between EMC1 and Rh1-apoprotein, Rh1-Gal4 driver was also used because the expression of EMC1::GFP was stronger in the photoreceptors. We added this sentence in the result section.

Reviewer #3

1) The authors show that EMC1::GFP does not co-immuno-precipitate with Rh1. Does calnexin co-IP with Rh1? I ask because the authors conclude (in the Discussion) that “ EMC is unlikely to be working in the calnexin cycle”, and it is difficult for me to understand why the authors have come to that conclusion.

Indeed, it is reported that Cnx is co-immunoprecipitated with Drosophila Rh1 (Rosenbaum, 2006).

Our result indicated that EMC1-GFP co-IPs with Cnx. If EMC-Cnx complex work for Rh1 in calnexin-cycle, physical interaction is expected between ER-accumulated Rh1 apoprotein and EMC-Cnx complex. However, EMC1::GFP does not co-IP with Rh1, suggesting EMC-Cnx complex does not function in the calnexin-cycle but Cnx does.

In addition to the calnexin cycle, Cnx is also know to recognizes nascent polypeptides co-translationally in the ER lumen (Chen, 1995). We think it is more likely EMC-Cnx complex functions in this process.

2) The authors report negative results with the ERAD component mutants, EDEM1, EDEM2 and VCP/+. I don't see the point of showing such negative data, as one cannot draw any conclusions. Perhaps the individual EDEM mutants do not show Rh1 phenotype as they are redundant (in mammals, EDEMs are known to be genetically redundant). Also, VCP's effect was assessed in a heterozygous condition, and a negative result here simply means that there is no dominant genetic effect. How about analyzing homozygous clones? Alternatively, since ERAD is not a major point of this work, the authors may want to take out this part, as it does not contribute to the overall story.

Regarding the redundancy for EDEM, it is shown that RNAi knockdown of EDEM1 increases aggregation of P23H folding-mutant of human rhodopsin (Kosmaoglou, 2009). Drosophila has only two EDEM orthologs, EDEM1 and EDEM2 in the genome. We used mutant clones lacking dPob, EDEM1 and EDEM2, but did not see Rh1 apoprotein accumulation in the ER.

Regarding the heterozygous usage for VCP, It is shown that The degradation of Rh1 intermediate in Rh1[P37H] folding-mutant is restored by the heterozygous mutation of TER9426-8 (Griciuc, 2010).

However, we also agree with the reviewer’s opinion: these negative data does not draw clear conclusions. Therefore, we will take out this part.

[Editors’ note: the author responses to the previous round of peer review follow.]

The reviewers noted the significance of your discovery of a role for a Drosophila member of the EMC complex in the maturation of rhodopsin. Your discovery that dPob/EMC3 acts upstream of ninaA in rhodopsin maturation was specifically credited with supporting a role for this member of the EMC complex in the early steps of the maturation of a multi-pass membrane protein. However, the expert reviewers were unanimous in their view that these finding do not add up to an advance of sufficient measure to merit publication in eLife.

It is possible that a different manuscript, for example one with further details on genetic lesions in other Drosophila EMC homologs or one that carried the biochemical analysis of the dPob/EMC3 lesion further would had received a warmer welcome at eLife. Unfortunately, the manuscript before us is too far off this mark to suggest specific experiments that would render it suitable for this journal.

Thus we are left with no choice but to return the manuscript to you in the hope that you will find the reviewers’ comments (appended below) of use as you prepare it for submission elsewhere.

We are pleased to read reviewer’s constructive criticism and suggestion, and tried to answer them with sincere. Our revised manuscript includes three sets of new data, and three minor changes in figures, based on reviewer’s suggestion. Based on our new data, we slightly shifted our conclusion from the original manuscript, but mostly the same. We believe now our conclusion, “EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors”, becomes more solid and reliable.

Three sets of new data are following:

1) In our original manuscript, we analyzed only a subunit of EMS, dPob/EMC3. However, in this revised manuscript, in a large scale screening of EMS induced mutants deficient in Rh1 expression, we identified only two mutants with dPob-like phenotype. These two mutants were carrying loss of function mutations of EMC subunits: one is on EMC1, and the other is on EMC8/9, which yeast lacks. We analyzed the accumulation of immature Rh1 and substrate specificities in these mutants. In both loss of function mutants for EMC1 and EMC8/9, immature Rh1 fails to accumulate in ER (Figure 4), and the expressions of multi-pass transmembrane proteins, but neither a secreted nor type-I, II and IV single-pass transmembrane proteins are greatly reduced (Figure 7). The substrate specificities shown in the deficiencies of EMC1 and EMC8/9 are exactly same as that in dPob deficiency. This is the first functional study of the additional subunits of EMC, which yeast lacks.

2) The absence of Rh1 apoprotein can be explained by the degradation of misfolded Rh1 in the EMC null mutants through the accelerated ERAD pathway. Thus, we investigated if mutations on ERAD components restore immature Rh1 in ER. However, unlike in the two folding mutants of Rh1 (Kang and Ryoo, 2009; Kosmaoglou et al., 2009; Griciuc et al., 2010), disturbance of ERAD activity did not restore expression of Rh1 in dPob/EMC3-deficient photoreceptors (Figure 9C, D). Together with the epistasis over ninaA, the higher susceptibility of the Rh1 to the ERAD pathway than that of the two folding mutants of Rh1, imply that the EMC complex is more likely to be involved in the earlier processes such as membrane integration or co-translational folding than in the folding of fully translated, membrane integrated Rh1-apoprotein.

3) We performed co-IP experiment using EMC1::GFP as a bait, because dPob::GFP is not functional. We could confirm EMC1::GFP interacts with dPob, but we failed to show significant interaction between EMC1::GFP and immature Rh1 apoprotein in the ER (Figure 5). This result also supports the EMC complex is likely involved in the early biogenesis for multi-pass membrane proteins than in the folding of fully translated, membrane integrated Rh1-apoprotein.

By this co-IP experiment, we also found EMC1::GFP interacts with calnexin (Cnx). It has been reported that the photoreceptors of an amorphic mutant of Cnx show complete loss of Rh1 apoprotein (Rosenbaum et al., 2006) just as we showed in dPob, EMC1 or EMC8/9 mutant. Moreover, both Cnx and EMC are epistatic to the mutant of the rhodopsin-specific chaperon NinaA, which accumulates Rh1 apoprotein in the ER. These results indicate that EMC and Cnx can work together in Rh1 biosynthetic cascade prior to NinaA works.

Three minor changes in figures are following:

1) We add Rh1 staining of WT, ninaAp263 mutant, dPobdelta4 mutant ommatidia (Figure 3B-D).

2) This revised manuscript includes the analysis of three single-pass transmembrane proteins and one double-pass transmembrane protein, which we did not work for the original manuscript (Figure 6F-H).

3) We removed Figure 9 in the original manuscript. We just described the results in the text.

Reviewer #1

[…] Other findings, such as the altered ER morphology in mutant tissue and the activation of the UPR are less informative and more anticipated by the yeast work.

Thus the crucial issue for the reviewers is to establish the strength of the experimental data and, importantly the degree to which it advances our understanding of the EMC's biological role. If the application of this Drosophila genetic system has provided strong evidence favoring a selective role for this EMC component in early biogenesis of multi-pass transmembrane proteins, then the paper would be of interest to a broad readership regardless of the degree to which these findings were anticipated by prior speculation. If, however, the additional experimental data derived from the Drosophila system were deemed merely incremental, the paper would be more suited for a specialist journal.

In this revised version of our paper, we investigated not only dPob/EMC3 deficiency, but also the deficiencies for other two subunits of EMC (see the answer for Reviewer#2 in detail). These two mutants were identified in a large scale screening of EMS-induced mutant deficient in Rh1 expression. Among 233 lines of Rh1-expression mutants, only two of them showed dPob-like phenotype (loss of Rh1 and NaK-ATPase but normal Eys expression), and these two were loss of function mutant of EMC subunits. Importantly, the deficiencies for three subunits of EMC gave the exactly same substrate specificity: they are essential for all of multi-pass transmembrane proteins, but not for a secreted protein or type-I, II and IV single-pass transmembrane proteins. Coincidence of the phenotypes in the deficiencies for three subunits of EMC provides the strong evidence favoring a selective role for this EMC component in early biogenesis of multi-pass transmembrane proteins.

Reviewer #2

[…] Although the authors have done a solid job in their characterization of dPob function in Drosophila, it appears to be an extension of the C. elegans work (Richard, 2013), but with more analysis of potential substrates. Moreover, this study does not address many of the pressing questions regarding the EMC complex. Does dPob's role in Drosophila reflect the entire EMC complex function? What is the mechanistic basis of the specificity of dPob towards multispan membrane proteins? Based on these grounds, whether the overall novelty and scope of this study justifies the publication of eLife can be subject to debate. Below are a few specific comments along these lines.

1) The authors had identified dPob through a previously reported genetic screen (Satoh, 2013). Were there other EMC homologs identified in the screen? If it is the case, the authors should highlight those results, as it would indicate that the observed effect is not an isolated function of dPob.

The previous screening among FRT-combined transposon insertion lines included only dPob/EMC3 among EMC subunits. The null mutant of dPob shows quite characteristic phenotype; no detectable protein expression of Rh1, and very weakened expression of other multiple-transmembrane domain proteins such as Na+K+-ATPase or TRP in the mosaic retina. We did not find any other mutant lines with such phenotype in the course of the mosaic screening among 546 insertional mutants described previously (Satoh et al., 2013). To explorer other mutants showing phenotypes similar to dPob null mutant, we examined a collection of 233 mutant lines deficient of Rh1 accumulation in photoreceptor rhabdomeres, obtained in an ongoing ethyl methanesulfonate (EMS) mutagenesis screening. Among them, only two lines, 665G and 008J showed dPob-like phenotype in the mean of distribution of Rh1 and Na+K+-ATPase in the mosaic retina. 665G and 008J turned out to be frame-shift and nonsense mutations of EMC1 and EMC8/9, respectively. Thus, we included the results of analysis for these mutations to our revised manuscript.

2) EMC genes are grouped because of their genetic interactions with each other in yeast. This study is potentially significant because the function of EMCs still remain poorly understood. Naturally, one must ask whether other EMC gene homologs show similar effects in rhodopsin maturation or not. The authors can examine this through in vivo RNAi, or better, by use of available mutant alleles.

We found the null mutants of EMC1 and EMC7/8 show the phenotype which is characteristic to the dPob null mutant. Mutants of other subunits of EMC were not available. We did not perform RNAi experiment of other EMCs because the phenotype expected in RNAi experiment will be hypomorphic, and we knew the hypomorphic mutant of dPob shows just reduced Rh1 expression. Previously, we have tried RNAi screening of genes required for the Rh1 expression, and found out that too many genes show reduced Rh1 expression when derived by GMR-Gal4, probably because of the off-target effect, and none of them were phenocopied by null allele of the genes. Contrary, when RNAi was derived by Rh1-Gal4, the genes known to be required for Rh1 transport, showed no phenotype. Altogether, in Drosophila photoreceptor, we think RNAi experiments will not provide phenotype specific enough to conclude something.

3) Does dPob physically interact with rhodopsin and other multipass membrane proteins? Immuno-precipitation experiments may be useful.

We performed co-IP experiment using EMC1::GFP as a bait, because dPob::GFP did not localize to the ER. We could confirm EMC1::GFP interacts with dPob, but we failed to show the stable interaction between EMC1::GFP and Rh1 apoprotein accumulated in the ER in the VA-condition.

In addition, we found the mutations of ERAD components fail to restore Rh1 immature in dPob mutant cells. From these two results with epistasis over ninaA, we now think EMC complex is more likely to be involved in the earlier processes such as membrane integration or co-translational folding than in the folding of fully-translated, membrane-integrated Rh1-apoprotein.

Interestingly, we found Calnexin (Cnx) binds to EMC1::GFP in the co-IP experiment. The photoreceptors of an amorphic mutant of Cnx show complete loss of Rh1 apoprotein (Rosenbaum, 2006) just as shown in dPob, EMC1 or EMC8/9 mutant. Moreover, both Cnx and EMC are epistatic to the mutant of the rhodopsin-specific chaperon NinaA, which accumulates Rh1 apoprotein in the ER. These results indicate that EMC and Cnx can work together in Rh1 biosynthetic cascade prior to NinaA works.

4) Is there any mechanistic insight as to how dPob would specifically recognize multipass membrane proteins? Although the authors show the dPob locus in Figure 1, they do not describe any domain structures in the gene. The fact that dPob specifically affects transmembrane domain is an intriguing phenomenon, but a mechanistic insight is lacking.

We now think EMC complex is more likely to be involved in the earlier processes such as membrane integration or co-translational folding, than in the folding of fully-translated, membrane-integrated Rh1-apoprotein (see above for the reason of this).

Most of the newly synthesized multi-pass membrane proteins are co-translationally integrated into the ER membrane through the translocon complex, and only one or two transmembrane helices can be stored in the translocon channel and the lateral gate. The helices associated with the translocon are displaced by the next set of newly synthesized transmembrane helices in the membrane-integration of multi-pass membrane proteins (Cymer, 2014; Rapoport, 2004). We assume that EMC may bind and stabilize the displaced helices, facilities the displacement, or involves to the integration, however, only sophisticated biochemistry with in vitro translation system can address the issue. We are applying for funding to start it, though we do not have enough resource to do it now.

5) Does dPob physically interact with rhodopsins and other targets? A simple immune-precipitation experiment should provide answers.

We answered to this question in point 3 above.

Reviewer #3

[…] This work is solid and interesting. It presents a good set of figures. I don't perceive the manuscript as outstanding because the actual function of dPob is inferred from correlations with highly complex steady-state effects on selected individual proteins or on global ER homeostasis. From an outstanding paper I would expect either a systematic and unbiased approach to identifying the substrates of the putative chaperone complex or a delineation of the features that it recognizes in its substrates or some direct evidence for the actual chaperone activity proposed.

Because of the limitation of our model system, we could not perform a systematic approach to identifying the substrates. Instead, we looked more substrates and we used more accurate topological classification for membrane proteins.

Now we do not think chaperon is the only possible function of EMC. We showed that weakening ERAD pathway does not suppress the loss of Rh1 by slowing down ERAD-dependent degradation. We also performed Co-IP with EMC1::GFP but did not detect stable binding with ER-accumulated Rh1 apoprotein. Our new results indicate it is more possible that EMC has some function on earlier steps than folding of fully translated protein, such as stabilizing translation, co-translational folding or membrane integration.

https://doi.org/10.7554/eLife.06306.014

Article and author information

Author details

  1. Takunori Satoh

    Graduate School of Integrated Arts and Science, Hiroshima University, Higashi-Hiroshima, Japan
    Contribution
    TS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Aya Ohba

    Graduate School of Integrated Arts and Science, Hiroshima University, Higashi-Hiroshima, Japan
    Contribution
    AO, Acquisition of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  3. Ziguang Liu

    Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences, Harbin, China
    Contribution
    ZL, Acquisition of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  4. Tsuyoshi Inagaki

    Graduate School of Integrated Arts and Science, Hiroshima University, Higashi-Hiroshima, Japan
    Contribution
    TI, Acquisition of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  5. Akiko K Satoh

    Graduate School of Integrated Arts and Science, Hiroshima University, Higashi-Hiroshima, Japan
    Contribution
    AKS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    aksatoh@hiroshima-u.ac.jp
    Competing interests
    The authors declare that no competing interests exist.

Funding

Japan Science and Technology Agency (JST) (PRESTO (Precursory Research for Embryonic Science and Technology) 25-J-J4215)

  • Akiko K Satoh

Japan Society for the Promotion of Science (JSPS) (21687005 KAKENHI)

  • Akiko K Satoh

Japan Society for the Promotion of Science (JSPS) (21113510 KAKENHI)

  • Akiko K Satoh

Japan Society for the Promotion of Science (JSPS) (30529037 KAKENHI)

  • Akiko K Satoh

The Naito Foundation (25-040920)

  • Akiko K Satoh

The Novartis Foundation (25-050421)

  • Akiko K Satoh

The Hayashi Memorial Foundation for Female Natural Scientists (25-051022)

  • Akiko K Satoh

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Drs U Tepass, C Montell, C Zuker, H Ryoo, and J Han who kindly provided fly stocks and reagents. We also thank the Bloomington Stock Center and the Drosophila Genetic Resource Center of the Kyoto Institute of Technology for fly stocks. This study was supported by grants from the Naito Foundation (25-040920), the Novartis Foundation (25-050421), the Hayashi Memorial Foundation for Female Natural Scientists (25-051022), PRESTO (25-J-J4215), and KAKENHI (21687005, 21113510, and 23113712) to ASK. This study was also supported by grants from the Global Centers of Excellence Program ‘Advanced Systems-Biology: Designing The Biological Function’ from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. Whole genome and targeted re-sequencing was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.

Reviewing Editor

  1. David Ron, Reviewing Editor, University of Cambridge, United Kingdom

Publication history

  1. Received: December 31, 2014
  2. Accepted: January 26, 2015
  3. Version of Record published: February 26, 2015 (version 1)

Copyright

© 2015, Satoh et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,194
    Page views
  • 302
    Downloads
  • 12
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Comments

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)