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SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes

  1. James P Zewe
  2. Rachel C Wills
  3. Sahana Sangappa
  4. Brady D Goulden
  5. Gerald RV Hammond  Is a corresponding author
  1. University of Pittsburgh School of Medicine, United States
Research Article
Cite as: eLife 2018;7:e35588 doi: 10.7554/eLife.35588
6 figures, 1 table and 1 additional file

Figures

Inhibition of SAC1 causes PtdIns4P accumulation in the ER.

(A) A soluble fragment of SAC1 (SAC1∆TMD) is inhibited by peroxide and bpV(HOpic). (B) SAC1 expression depletes PM PtdIns4P. COS-7 cells transfected with GFP-P4M and either FKBP-mCherry (Ctrl), SAC1∆TMD-FKBP-mCherry or catalytically inactive SAC1∆TMD/C389S-FKBP-mCherry were imaged live by confocal microscopy. Representative images are shown (bar = 10 µm). The graph shows P4M intensity at the plasma membrane (defined by CellMask deep red dye) normalized to total cell intensity; box and whisker plot shows quartiles and 5–95 percentiles of 90 cells from three independent experiments. P values derive from Dunn’s multiple comparison test compared to Ctrl after a Kruskal-Wallis test (p<10–4). (C) Peroxide and bpV(HOpic) inhibit SAC1 in live cells. COS-7 cells were transfected with P4M and SAC1∆TMD as in B and imaged by time-lapse confocal microscopy. 500 µM peroxide or 10 µM bpV(HOpic) were added at time 0. P4M intensity was quantified as in B. Data are means ± s.e. of 44 or 45 cells from four independent experiments. Scale bar = 10 µm. (D) Predicted PtdIns4P accumulation for ‘cis’ and ‘trans’ operation of SAC1. (E) Peroxide does not disrupt ORP5 localization at ER-PM MCS. Images show TIRF images of COS-7 cells expressing GFP-ORP5 at the indicated times. Traces are means with s.e. shaded for 31–32 cells from three independent experiments. (F–G) SAC1 inhibitors cause PtdIns4P accumulation in the ER. Time-lapse images of representative COS-7 cells expressing GFP-P4M (F) or GFP-P4C (G) and treated with inhibitors at time 0. The insets are 10 µm squares, and are expanded at right and show PtdIns4P accumulation relative to a co-expressed ER marker, iRFP-Sec61β. Graphs show P4M intensity at the ER (defined by iRFP-Sec61β) normalized to total cell intensity; data are means ± s.e. of 38–41 (F) or 29–30 (G) cells from three (G) or four (F) independent experiments.

https://doi.org/10.7554/eLife.35588.002
Localization of SAC1 relative to ER-PM MCS and ER proteins.

(A) Strategy for tagging endogenous SAC1: a guide RNA is complexed with Cas9 protein and electroplated into HEK-293A cells with a short single-stranded homology-directed repair (HDR) template. This adds a short tag encoding the 11th strand of the GFP beta barrel. When expressed, this strand assembles with co-expressed GFP1-10 to make functional GFP. (B) Specificity of genomic tagging. 293A cells stably over-expressing GFP1-10 and edited with the indicated GFP11 tags were genotyped with GFP11 specific forward primers and a gene-specific reverse primer located ~200 bp downstream in exon 1. (C) Confocal images of GFP11 gene edited cells co-expressing mKo-Manosidase II as a cis/medial Golgi marker, or mCherry-VAPB as an ER marker. (D) E-Syt1 shows enrichment at the PM relative to SAC1 and Sec61β. Cells were imaged in both TIRF and epi-illumination, and the fluorescence intensity ratio of the two images was calculated. Boxes represent quartiles, whiskers 5–95 percentile. P values are from Dunn’s Multiple Comparisons following a Kruskall-Wallis test (p<10–4). Data are from 180 (E-Syt1), 234 (SAC1) or 246 (Sec61β) cells imaged across five independent experiments. Insets = 10 µm. (E) Expressed SAC1 is not enriched at ER-PM MCS in COS-7 cells. TIRF images of COS-7 cells transfected for 24 hr with the indicated GFP-tagged plasmid and mCherry-MAPPER to label ER-PM MCS along with iRFP-Sec61β to label total ER. Scale bar = 10 µm. The MCS index is the ‘difference of differences’ between GFP and iRFP-Sec61β as well as GFP and MAPPER signals. P values are from Dunn's Multiple Comparison test relative to GFP-Sec61β, run as a post-hoc to a Kruskal-Wallis test (p<10–4). Box and whiskers are quartiles with 10–90 percentiles of 90 (Sec61β), 92 (Calreticulin), 91 (SAC1), 93 (MAPPER) or 92 (E-Syt2, ORP5) cells imaged across three independent experiments.

https://doi.org/10.7554/eLife.35588.008
Recruitment of proteins to ER-PM MCS.

(A) Transfected SAC1 does not dynamically re-distribute to ER-PM MCS in COS-7 cells. Time-lapse TIRF microscopy of COS-7 cells transfected with the indicated GFP-tagged proteins for 6–7 hr. Cells were stimulated with 100 µM ATP as indicated. Insets = 10 µm. The traces at right show ∆(Ft/Fpre) and are means ± s.e. of 30 (Sec61β, SAC1, Calreticulin), 27 (STIM1), 29 (ESyt1) or 20 (Nir2) cells imaged across three independent experiments. (B) Gene edited alleles do not perturb calcium signals. Edited 293AGFP1-10 cells were loaded with Fura-red and the ratio of fluorescence intensity with respect to 405 and 488 nm excitation was measured. Cells were stimulated with carbachol (CCh) at 30 s to activate phospholipase C signaling. Data are grand means of four experiments (shaded regions represent s.e.). The P value represents results of a two-way ANOVA comparing cell lines. (C) Endogenous SAC1 does not recruit to ER-PM contact sites in 293AGFP1-10 cells. Images show representative gene-edited cells at the indicated times during time-lapse TIRF imaging. Carbachol was added to stimulate phospholipase C signaling at time 0. Images are averages of 5 frames acquired over 10 s to improve signal to noise. Traces represent mean change in fluorescence intensity (normalized to pre-stimulation levels) with s.e. of 40 (E-Syt1), 38 (SAC1) or 37 (Sec61β) cells imaged across five independent experiments.

https://doi.org/10.7554/eLife.35588.011
SAC1 is much more active at the PM in ‘cis’.

(A) Strategy to recruit SAC1 to the PM in ‘cis’ or ‘trans’ using the FRB/FKBP12 heterodimerization system. (B) PM PtdIns4P is still detectable at the PM with P4M × 2 after transfection with SAC1∆TMD. COS-7 cells transfected with GFP-P4M and either FKBP-mCherry (Ctrl), SAC1∆TMD-FKBP-mCherry or catalytically inactive SAC1∆TMD/C389S were imaged live by confocal microscopy. Representative images are shown (bar = 20 µm). The graph shows P4M intensity at the plasma membrane (defined by CellMask deep red dye) normalized to total cell intensity; box and whisker plot shows quartiles and 5–95 percentiles of 90 cells from three independent experiments. P values derive from Dunn's multiple comparison test compared to Ctrl after a Kruskal-Wallis test (p<10–4). (C) Recruitment of SAC1 to the PM in ‘cis’ is far more effective in depleting PtdIns4P than it is in ‘trans’. TIRF images of COS-7 cells transfected with a Lyn11-FRB-iRFP PM recruiter, the indicated mCherry-tagged SAC1-FKBP or FKBP-SAC1, and GFP-P4M × 2. Graphs show means ± s.e. Images are representative of n cells, x independent experiments: 57, 6 (FKBP-SAC1); 41, 4 (FKBP-SAC1C389S); 28, 3 (SAC1-FKBP); 30, 3 (SAC1-FKBP); 57, 6 (SAC1-FKBP); 36, 4 (SAC1-FKBP); 26, 3 (FKBP-SAC1); 29, 3 (SAC1∆452-587). Inset graphs show the raw change in signal intensity for the mCherry-FKBP tagged SAC1 chimeras. Images of GFP-P4M × 2 are normalized to the mean pre-stimulation pixel intensity, that is Ft/Fpre with the color coding reflected in the graph y-axis. Scale bar = 20 µm.

https://doi.org/10.7554/eLife.35588.015
An extended helical linker confers ‘trans’ activity to SAC1.

(A) Helical linkers (HL) added to FKBP-SAC1 at the end of the first transmembrane domain. Each helical repeat consists of the amino acids EAAAR, expected to form a helix approximately 0.75 nm long. (B) TIRF imaging of PtdIns4P before and after direct recruitment of SAC1 to ER-PM MCS. TIRF images of COS-7 cells transfected with a Lyn11-FRB-iRFP PM recruiter, the indicated mCherry-tagged SAC1-FKBP and GFP-P4M × 2. Images are representative of 30 cells from three independent experiments. Images of GFP-P4M × 2 are normalized to the mean pre-stimulation pixel intensity, that is Ft/Fpre with the color coding reflected in the graph y-axis of D. Scale bar = 20 µm. (C) Helical linkers do not impair recruitment efficiency of FKBP-SAC1. (D) FKBP-SAC1-HLx8 and -HLx10 have ‘trans’ activity. Graphs in C and D show fluorescence intensity in the TIRF footprint of each cell for mCherry-tagged FKBP-SAC1 or GFP-tagged P4M × 2, respectively. Data are means ± s.e., 30 cells for all except WT, with 57 cells. Data for the wild-type FKBP-SAC1 is re-plotted from Figure 4.

https://doi.org/10.7554/eLife.35588.019
An extended helical linker is required for ‘trans’ activity of SAC1 at induced ER-PM MCS.

(A) Induction of artificial ER-PM MCS using rapamycin-induced dimerization of PM Lyn11-FRB and ER FKBP-CYB5Atail. (B) Over-expression of E-Syt2 does not deplete PtdIns4P. COS-7 cells over-expressing GFP-tagged E-Syt2, ORP5 along with mCherry-P4M × 2; scale bar = 10 µm. Graph shows P4M intensity at the plasma membrane (defined by CellMask deep red dye) normalized to total cell intensity; box and whisker plot shows quartiles and 5–95 percentiles of 89–90 cells from three independent experiments. P values derive from Dunn’s multiple comparison test compared to Ctrl after a Kruskal-Wallis test (p<10–4). (C) FKBP-CYB5tail induces narrower contact sites than those occupied by E-Syt2 or ORP5. COS-7 cells expressing the indicated GFP-fusion protein, Lyn11-FRB-iRFP or mCherry-FKBP-CYB5tail (not shown), dimerization induced with Rapa as indicated. Graph shows the fraction of induced contact sites occupied by GFP-fluorescence after 5 min of rapa treatment; box and whisker plot shows quartiles and 5–95 percentiles of 14–19 cells from four independent experiments. P values derive from Dunn’s multiple comparison test compared to Ctrl after a Kruskal-Wallis test (p<10–4). (D) An extended helical linker is required for robust ‘trans’ activity of SAC1 at ER-PM MCS. Images of TagBFP2-tagged FKBP-CYB5 and GFP-P4M × 2 in COS-7 cells co-transfected with iRFP-tagged Lyn11-FRB and the indicated mCherry-tagged SAC1 construct, or mCherry alone as control. Images of GFP-P4M × 2 are normalized to the mean pre-stimulation pixel intensity, that is Ft/Fpre with the color coding reflected in the graph y-axis. Scale bar = 20 µm. Graphs show the fluorescence intensity of GFP-P4M × 2 in the TIRF footprint of each cell (means ± s.e., 29–30 cells from three independent experiments) normalized to the mean pre-stimulation level (Fpre).

https://doi.org/10.7554/eLife.35588.022

Tables

Table 1
Plasmids used in this study.

Genes are human unless otherwise stated

https://doi.org/10.7554/eLife.35588.026
PlasmidBackboneInsertRef
 APX1-GFP1-10APX1super-folder GFPThis study
 CDV-hyPBasepiggpiggyBAC transposase(Yusa et al., 2011)
 NES-EGFP-P4M × 1pEGFP-C1X.leavis map2k1.L(32-44):EGFP:L. pneumophila SidM(546-647)This study
 FKBP-mCherrypmCherry-N1FKBP1A(isoform a, 3–108):mCherryThis study
 SAC1∆TMD-FKBP-mCherrypmCherry-N1SACM1L(1-521):FKBP1A(3-108):mCherryThis study
 SAC1C389S∆TMD-FKBP-mCherrypmCherry-N1SACM1L(C389S; 1–521):FKBP1A(3-108):mCherryThis study
 iRFP-Sec61βpiRFP-C1iRFP:SEC61BThis study
 mKO-ManIIpmKO-N1Kusabira Orange 2:Man2a(1-102)Tamas Balla
 mCherry-VAPBpmCherry-C1mCherry:VAPBThis study
 mCherry-MAPPERpmCherry-C1mCherry:MAPPER(Chang et al., 2013)
 EGFP-MAPPERpEGFP-C1EGFP:MAPPER(Chang et al., 2013)
 GFP-ORP5pEGFP-C1EGFP:OSBPL5(isoform a)(Sohn et al., 2016)
 GFP-E-Syt2pEGFP-C1EGFP:ESYT2(Giordano et al., 2013) Addgene plasmid #66831
 EGFP-SAC1pEGFP-C1EGFP:SACM1L(Sohn et al., 2016)
 mEmerald-N16-CalreticulinpmEmerald-N1mEmerald:CALRMichael Davidson (Addgene plasmid #54023)
 GFP-Sec61βpAcGFP-C1-Sec61βAequorea coerulescens GFP:SEC61B(Voeltz et al., 2006) Addgene plasmid #15108
 EGFP-E-Syt1pEGFP-C1EGFP-ESYT1(Giordano et al., 2013) Addgene plasmid #66830
 EGFP-STIM1pEGFP-C1STIM1(isoform 1 1–22):EGFP:STIM1(23–791)(Várnai et al., 2007)
 EGFP-Nir2pEGFP-N1EGFP:PITPNM1(isoform 2)(Kim et al., 2015)
 EGFP-P4M × 2pEGFP-C1EGFP:L. pneumophila SidM(546-647):SidM(546-647)(Hammond et al., 2014)
 Lyn11-FRB-iRFPpiRFP-N1LYN(1-11):MTOR(2021–2113):iRFP(Hammond et al., 2014)
 mCherry-FKBPpmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG(Hammond et al., 2014)
 mCherry-FKBP-SAC1pmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1LThis study
 mCherry-FKBP-SAC1C389SpmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1LC389SThis study
 mCherry-SAC1-FKBPpmCherry-C1mCherry:SACM1L:FKBP1A(3-108)This study
 mCherry-SAC1C389S-FKBPpmCherry-C1mCherry:SACM1LC389S:FKBP(3-108)This study
 SAC1∆452-587-FKBP-mCherrypmCherry-N1SACM1L(1-451):FKBP1A(3-108):mCherryThis study
 mCherry-FKBP-SAC1∆TMDpmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L(1-521)This study
 mCherry-FKBP-SAC1-HLx2pmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG::SACM1L(1-520):[EAAAR]2:SACM1L(521-587)This study
 mCherry-FKBP-SAC1-HLx4pmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG::SACM1L(1-520):[EAAAR]4:SACM1L(521-587)This study
 mCherry-FKBP-SAC1-HLx6pmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L(1-520):[EAAAR]6:SACM1L(521-587)This study
 mCherry-FKBP-SAC1-HLx8pmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L(1-520):[EAAAR]8:SACM1L(521-587)This study
 mCherry-FKBP-SAC1-HLx10pmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L(1-520):[EAAAR]10:SACM1L(521-587)This study
 mCherry-FKBP-SAC1C389S-HLx8pmCherry-C1mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1LC389S(1-520):[EAAAR]8:SACM1L(521-587)This study
 mCherrypmCherry-C1mCherry(Hammond et al., 2014)
 mCherry-SAC1pmCherry-C1mCherry:SAC1ML(Sohn et al., 2016)
 mCherry-SAC1-HLx8pmCherry-C1mCherryLSACM1L(1-520):[EAAAR]8:SACM1L(521-587)This study
 mCherry-SAC1C389S-HLx8pmCherry-C1mCherry:SACM1LC389S(1-520):[EAAAR]8:SACM1L(521-587)This study
 mTagBFP2-FKBP-CYB5AtailpmTagBFP2-C1mTagBFP2:FKBP1A(3-108):[GGSA]4GG:CYB5A(100-134)This study

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