SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes
- University of Pittsburgh School of Medicine, United States
Figures
Figure 1
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.
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Figure 1—source data 1
Data for panel 1B.
- https://doi.org/10.7554/eLife.35588.003
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Figure 1—source data 2
Data for panel 1C.
- https://doi.org/10.7554/eLife.35588.004
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Figure 1—source data 3
Data for panel 1E.
- https://doi.org/10.7554/eLife.35588.005
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Figure 1—source data 4
Data for panel 1F.
- https://doi.org/10.7554/eLife.35588.006
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Figure 1—source data 5
Data for panel 1G.
- https://doi.org/10.7554/eLife.35588.007
Figure 2
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.
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Figure 2—source data 1
Data for panel 2D.
- https://doi.org/10.7554/eLife.35588.009
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Figure 2—source data 2
Data for panel 2E.
- https://doi.org/10.7554/eLife.35588.010
Figure 3
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.
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Figure 3—source data 1
Data for panel 3A.
- https://doi.org/10.7554/eLife.35588.012
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Figure 3—source data 2
Data for panel 3B.
- https://doi.org/10.7554/eLife.35588.013
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Figure 3—source data 3
Data for panel 3C.
- https://doi.org/10.7554/eLife.35588.014
Figure 4
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.
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Figure 4—source data 1
Data for panel 4B.
- https://doi.org/10.7554/eLife.35588.016
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Figure 4—source data 2
Data for panel 4C.
- https://doi.org/10.7554/eLife.35588.017
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Figure 4—source data 3
Data for panel 4C (insets).
- https://doi.org/10.7554/eLife.35588.018
Figure 5
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.
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Figure 5—source data 1
Data for panel 5C.
- https://doi.org/10.7554/eLife.35588.020
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Figure 5—source data 2
Data for panel 5D.
- https://doi.org/10.7554/eLife.35588.021
Figure 6
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).
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Figure 6—source data 1
Data for panel 6B.
- https://doi.org/10.7554/eLife.35588.023
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Figure 6—source data 2
Data for panel 6C.
- https://doi.org/10.7554/eLife.35588.024
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Figure 6—source data 3
Data for panel 6D.
- https://doi.org/10.7554/eLife.35588.025
Author response image 1
Effects of SAC1 inhibition (with 500 µM peroxide) on PtdSer probe Lact-C2.
(A) COS-7 transfected with mCherry-Lact-C2 were treated with vehicle or 500 µM DMSO during imaging by TIRF microscopy. Data are means ± s.e. of 32 cells from two independent experiments. P value is from a two-way repeated measures ANOVA. (B) mCherry-Lact-C2 and (C) TagBFP2-P4M were co-transfected into the same cells with iRFP-Sec61β (as an ER marker) and either GFP-ORP5 or GFP-calreticulin as control. Cells were imaged by confocal microscopy and assimilation of the probes fluorescence in the ER was measured. Data are means ± s.e. of 32-35 cells from three independent experiments. P values are derived from Sidak’s multiple comparison test after two-way repeated measures ANOVA (P < 10–4 for both).
Author response image 2
Dissociation of ePH-ORP5 and -ORP8 by PtdIns4P or PtdIns(4,5)P2 depletion.
COS-7 cells were transfected with GFP-tagged ePH probes, Lyn11-FRB-iRFP recruiter and mCherry-FKBP-SAC1∆TMD or -INPP5E as indicated, and imaged by TIRF microscopy. Recruitment of enzymes was induced by rapamycin addition at time 0. Data are means ± s.e. of 6-10 cells from a single experiment.
Author response image 3
CRISPR/Cas9mediated acute kick-out of SAC1.
(A) Western Blot of duplicate lysates from CRISPR/Cas9 plasmid transfected cells (selected with puromycin) after 72 h of knock down were probes with both SAC1 antisera and monoclonal anti-tubulin antibody DM1A. One sample was sonicated after lysis. The boxed region in blots is expanded in (B). Images of GFP-P4C (C) or GFPePH-ORP8 (D) from the 1st (1-25th percentile) or 4th (75-100th percentile) cohort of control or CRISPR/Cas9 plasmid transfected cells. The Box plots show mean and interquartile range, and the whiskers the list and 4th quartiles of 29 (C) or 25-32 (D) cells from a single experiment. Intensity at the PM was measured using a CellMask-derived mask as described in the manuscript.
Tables
Table 1
Plasmids used in this study.
Genes are human unless otherwise stated
| Plasmid | Backbone | Insert | Ref |
|---|---|---|---|
| APX1-GFP1-10 | APX1 | super-folder GFP | This study |
| CDV-hyPBase | pigg | piggyBAC transposase | (Yusa et al., 2011) |
| NES-EGFP-P4M × 1 | pEGFP-C1 | X.leavis map2k1.L(32-44):EGFP:L. pneumophila SidM(546-647) | This study |
| FKBP-mCherry | pmCherry-N1 | FKBP1A(isoform a, 3–108):mCherry | This study |
| SAC1∆TMD-FKBP-mCherry | pmCherry-N1 | SACM1L(1-521):FKBP1A(3-108):mCherry | This study |
| SAC1C389S∆TMD-FKBP-mCherry | pmCherry-N1 | SACM1L(C389S; 1–521):FKBP1A(3-108):mCherry | This study |
| iRFP-Sec61β | piRFP-C1 | iRFP:SEC61B | This study |
| mKO-ManII | pmKO-N1 | Kusabira Orange 2:Man2a(1-102) | Tamas Balla |
| mCherry-VAPB | pmCherry-C1 | mCherry:VAPB | This study |
| mCherry-MAPPER | pmCherry-C1 | mCherry:MAPPER | (Chang et al., 2013) |
| EGFP-MAPPER | pEGFP-C1 | EGFP:MAPPER | (Chang et al., 2013) |
| GFP-ORP5 | pEGFP-C1 | EGFP:OSBPL5(isoform a) | (Sohn et al., 2016) |
| GFP-E-Syt2 | pEGFP-C1 | EGFP:ESYT2 | (Giordano et al., 2013) Addgene plasmid #66831 |
| EGFP-SAC1 | pEGFP-C1 | EGFP:SACM1L | (Sohn et al., 2016) |
| mEmerald-N16-Calreticulin | pmEmerald-N1 | mEmerald:CALR | Michael Davidson (Addgene plasmid #54023) |
| GFP-Sec61β | pAcGFP-C1-Sec61β | Aequorea coerulescens GFP:SEC61B | (Voeltz et al., 2006) Addgene plasmid #15108 |
| EGFP-E-Syt1 | pEGFP-C1 | EGFP-ESYT1 | (Giordano et al., 2013) Addgene plasmid #66830 |
| EGFP-STIM1 | pEGFP-C1 | STIM1(isoform 1 1–22):EGFP:STIM1(23–791) | (Várnai et al., 2007) |
| EGFP-Nir2 | pEGFP-N1 | EGFP:PITPNM1(isoform 2) | (Kim et al., 2015) |
| EGFP-P4M × 2 | pEGFP-C1 | EGFP:L. pneumophila SidM(546-647):SidM(546-647) | (Hammond et al., 2014) |
| Lyn11-FRB-iRFP | piRFP-N1 | LYN(1-11):MTOR(2021–2113):iRFP | (Hammond et al., 2014) |
| mCherry-FKBP | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG | (Hammond et al., 2014) |
| mCherry-FKBP-SAC1 | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L | This study |
| mCherry-FKBP-SAC1C389S | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1LC389S | This study |
| mCherry-SAC1-FKBP | pmCherry-C1 | mCherry:SACM1L:FKBP1A(3-108) | This study |
| mCherry-SAC1C389S-FKBP | pmCherry-C1 | mCherry:SACM1LC389S:FKBP(3-108) | This study |
| SAC1∆452-587-FKBP-mCherry | pmCherry-N1 | SACM1L(1-451):FKBP1A(3-108):mCherry | This study |
| mCherry-FKBP-SAC1∆TMD | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L(1-521) | This study |
| mCherry-FKBP-SAC1-HLx2 | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG::SACM1L(1-520):[EAAAR]2:SACM1L(521-587) | This study |
| mCherry-FKBP-SAC1-HLx4 | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG::SACM1L(1-520):[EAAAR]4:SACM1L(521-587) | This study |
| mCherry-FKBP-SAC1-HLx6 | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L(1-520):[EAAAR]6:SACM1L(521-587) | This study |
| mCherry-FKBP-SAC1-HLx8 | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L(1-520):[EAAAR]8:SACM1L(521-587) | This study |
| mCherry-FKBP-SAC1-HLx10 | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1L(1-520):[EAAAR]10:SACM1L(521-587) | This study |
| mCherry-FKBP-SAC1C389S-HLx8 | pmCherry-C1 | mCherry:FKBP1A(3-108):[GGSA]4GG:SACM1LC389S(1-520):[EAAAR]8:SACM1L(521-587) | This study |
| mCherry | pmCherry-C1 | mCherry | (Hammond et al., 2014) |
| mCherry-SAC1 | pmCherry-C1 | mCherry:SAC1ML | (Sohn et al., 2016) |
| mCherry-SAC1-HLx8 | pmCherry-C1 | mCherryLSACM1L(1-520):[EAAAR]8:SACM1L(521-587) | This study |
| mCherry-SAC1C389S-HLx8 | pmCherry-C1 | mCherry:SACM1LC389S(1-520):[EAAAR]8:SACM1L(521-587) | This study |
| mTagBFP2-FKBP-CYB5Atail | pmTagBFP2-C1 | mTagBFP2:FKBP1A(3-108):[GGSA]4GG:CYB5A(100-134) | This study |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.35588.027
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SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes
eLife 7:e35588.
https://doi.org/10.7554/eLife.35588
