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, 58 (3), 553-562

Phosphatidylserine Synthesis at Membrane Contact Sites Promotes Its Transport Out of the ER

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Phosphatidylserine Synthesis at Membrane Contact Sites Promotes Its Transport Out of the ER

Muthukumar Kannan et al. J Lipid Res.

Abstract

Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments.

Keywords: endoplasmic reticulum; lipid biochemistry; lipid transfer proteins; lipid transport; mitochondria; phosphatidylethanolamine; phosphatidylserine synthase; phospholipids/trafficking.

Figures

Fig. 1.
Fig. 1.
Localization and activity of PssA fusions used to study ER to mitochondria PS transport. A: Pathways of PC production in S. cerevisiae. Enzymes from yeast are in blue and red is used for E. coli. PC production by the Kennedy pathway requires exogenous choline. Multiple arrowheads indicate multiple steps. B: Diagram of PssA fusion proteins used to investigate PS transport to mitochondria. C: Growing cells expressing the indicated fusion proteins were visualized live. H2B-mCherry is a nuclear marker and Mdm12-mCherry is part of the ERMES complex. Scale bar, 5 μm. D: PS synthase activities of lysates from the indicated strains (mean ± SD, n = 3 independent experiments). E: Cells were labeled with [3H]serine and the amount of [3H]PS produced in 30 min was determined (mean ± SD, n = 3 independent experiments). F: The indicated strains were grown in SC with choline to mid-logarithmic growth phase, washed, and serial dilutions were spotted onto plates that either did or did not contain choline. The plates were incubated for 2 days at 30°C.
Fig. 2.
Fig. 2.
PS synthesis at ER-mitochondria contacts increases PS transport to mitochondria. A: The indicated strains were labeled with [3H]serine for 30 min and the percentage of [3H]PS converted to [3H]PE was determined (mean ± SD, n  =  3 independent experiments). *P < 0.05, independent two-tailed t-test. Raw data used to calculate percent of [3H]PS converted to [3H]PE is in supplemental Fig. S2A, B. B, C: Steady state levels of phospholipids in whole cells (B) and mitochondria (C) from the indicated strains grown in SG with choline (mean ± SD, n  =  3 independent experiments). *P < 0.05, independent two-tailed t-test. PI, phosphatidylinositol; PA, phosphatidic acid; CL, cardiolipin.
Fig. 3.
Fig. 3.
Increasing ER-mitochondria contacts increases PS transport to mitochondria. A–E: WT cells expressing empty vector (A) or ChiMERA (B–E) were visualized by EM. Boxed regions are shown in higher magnification. ER-mitochondria contact is highlighted in pink in the inset of the right panel of (A). Yellow arrows denote ER membrane. Green arrowheads indicate contact sites between the ER and mitochondria. The blue arrow denotes plasma membrane. M, mitochondria; V, vacuole; N, nucleus; PM, plasma membrane; CW, cell wall. F: Quantification of length of contact between ER and mitochondria for experiments in (A–E). The top panel shows the average ER-mitochondria ratio and the lower panel shows the average length of contacts (mean ± SD, n  =  15 individual cells). The ER-mitochondria ratio was determined by measuring the length of contacts between the ER and mitochondria divided by the length of mitochondrial perimeter in each image. ***P < 0.005, independent two-tailed t-test. G: The indicated strains were labeled with [3H]serine for 30 min and the percentage of [3H]PS converted to [3H]PE was determined (mean ± SD, n  =  3 independent experiments). *P < 0.05, independent two-tailed t-test. Raw data used to calculate percent of [3H]PS converted to [3H]PE is in supplemental Fig. S2C, D.
Fig. 4.
Fig. 4.
PS synthesis at ER-endosome contacts increases PS transport to endosomes. A: Growing cells expressing FFAT-GFP-PssA and the ER marker, ds-Red-HDEL, were visualized live. Scale bar = 5 μm. B: PS synthase activities of lysates from the indicated strains (mean ± SD, n = 3 independent experiments). C: The indicated strains were labeled with [3H]serine for 30 min and the percentage of [3H]PS converted to [3H]PE was determined (mean ± SD, n  =  3 independent experiments). *P < 0.05, independent two-tailed t-test. Raw data used to calculate percent of [3H]PS converted to [3H]PE is in supplemental Fig. S2E, F.
Fig. 5.
Fig. 5.
PS inhibits Cho1p. PS synthase activities of lysates from WT W303 cells with the indicated amounts of added PE or PS (mean ± SD, n = 3 independent experiments).

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