Lysophospholipids Facilitate COPII Vesicle Formation

Abstract

Coat protein complex II (COPII) proteins form vesicles from the endoplasmic reticulum to export cargo molecules to the Golgi apparatus. Among the many proteins involved in this process, Sec12 is a key regulator, functioning as the guanosine diphosphate (GDP) exchange factor for Sar1p, the small guanosine triphosphatase (GTPase) that initiates COPII assembly. Here we show that overexpression of phospholipase B3 in the thermosensitive sec12-4 mutant partially restores growth and protein transport at non-permissive temperatures. Lipidomics analyses of these cells show a higher content of lysophosphatidylinositol (lysoPI), consistent with the lipid specificity of PLB3. Furthermore, we show that lysoPI is specifically enriched in COPII vesicles isolated from in vitro budding assays. As these results suggested that lysophospholipids could facilitate budding under conditions of defective COPII coat dynamics, we reconstituted COPII binding onto giant liposomes with purified proteins and showed that lysoPI decreases membrane rigidity and enhances COPII recruitment to liposomes. Our results support a mechanical facilitation of COPII budding by lysophospholipids.

Keywords: COPII; budding assays; lysolipids; membrane curvature; membrane rigidity; phospholipase; vesicular transport.

Graphical abstract

Figure 1

Overexpression of PLB3 Rescues sec12-4 Mutant Phenotypes (A) Overexpression of PLB3 rescues temperature-sensitive growth defect of sec12-4 mutant. Five-fold serial dilution of 1 optical density 600 (OD₆₀₀)/mL of yeast culture was spotted onto YPD plates and were incubated at specified temperatures for 3 days. (B) Relative lysoPI species amount over the total of lysophospholipids in several strains. Usual lipid yield for cell extracts was 6 mmol. Error bars represent SD. t test values are significant according to ^∗p ≤ 0.05 and ^∗∗∗p ≤ 0.001. (C) Overexpression of PLB3 prevents ERES relocalization caused by sec12-4 at 30°C. Strains expressing SEC13-GFP were used to visualize the ERES at 24°C and 30°C. The scale bars represent 5 μm. (D) ERES per cell were counted and averaged from randomly chosen cells for each strain (wild-type [WT], n = 10; sec12-4, n = 13; WT+pPLB3, n = 15; sec12-4 +pPLB3, n = 13). Error bars represent SD. See also Figures S1 and S2 and Table S1.

Figure 2

ER Membranes and In Vitro Generated COPII Vesicles Are Enriched in Lysophospholipids Profile of major glycerophospholipid classes and their lyso counterpart measured by mass spectrometry (mol %). Error bars indicate SD unless noted different. (A) Lipid profile of wild-type yeast (median of n = 5) compared to the profile of microsomes (median of n = 8); ^∗p ≤ 0.05. (B) Lipid profiles of microsomes before (median of n = 8) and after (median of n = 12) budding reaction compared to the median lipid profile of COPII vesicles (median of n = 9). t test values are significant according to ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗p ≤ 0.001. (C) Ratios of COPII vesicle lipids versus microsomes before and after budding reaction. The mol % for each lipid type in vesicles is divided by the mol % of the microsome batch or microsomes post-budding from the same reaction used for that experiment; median represented in red with SEM. LPL, lysophospholipid. Usual lipid yield for cell extracts was 3.5 mmol and for microsomes was 0.5 mmol per reaction. Usual lipid yield for vesicles was 13 μmol after removing background. See also Figure S3 and Table S1.

Figure 3

LysoPI Increases the Binding of COPII to Membrane Surface and Lowers Bending Rigidity (A) Confocal images of COPII proteins binding to giant liposomes. GUVs were made of 36.5% DOPC, 20% DOPE, 30% soy PI, 8% DOPS, 5% DOPA, and 0.05% fluorescent rhodamine-PE. To test the effect of lysophospholipids, a similar mix, including 10% lysoPI, was added over the final weight of lipids and used in similar conditions. COPII proteins were mixed in a separate tube (Sar1 2.38 μM, Sec23/24 320 nM, and Sec13/31 562 nM) and incubated with GMP-PNP and EDTA to facilitate nucleotide exchange. After this short incubation, the protein mix was pipetted into the chamber with GUVs. Time-lapse images were taken for each GUV, 5 frames per second, 100 frames per channel. In total, four conditions were tested: with or without lysoPI combined with or without GMP-PNP. Scale bar, 10 μm. (B) After time averaging, the resulting GUV image was linearized and the intensity profile across the membrane was plotted. Background was subtracted and normalized the signal from inside the GUV to 0. (C) Intensity profiles obtained from the procedure shown in (B) for different conditions tested. Each profile is the average of five GUV profiles. Error bars represent SEM. (D) Scheme of experimental setup. An aspiration micropipette (left down) is used to set tension by aspirating a tongue from the GUV. A microbead trapped by an optical tweezer (right) is used to pull a nanotube from the GUV. A second micropipette (left up) injects lysoPI locally. (E) Linear variation of the force squared f² (N²) required to pull a tube from a vesicle as a function of membrane tension σ (N.m⁻¹) applied by the aspiration micropipette. Line slopes are proportional to the bending rigidity κ. Measurements were done on the same GUV before lysoPI was injected and afterward. (F) Average bending rigidity before and after injection of lysoPI (n = 5). Error bars represent SD. (G) Time-lapse of lysoPI injection (120 μM) in the vicinity of a GUV aspirated within a micropipette. Upon injection of lysoPI, a force drop is detected (not shown) and the aspiration tongue elongates until it stabilizes. The scale bar represents 10 μm. See also Videos S1, S2, S3, and S4.

Figure 4

Full COPII Coat Is Required for Optimal Binding to Membrane Surface (A) Averaged intensity profiles of Sar1 and Sec13/31 membrane binding with different lipid composition and conditions omitting Sec23/24 or with full COPII coats. Intensity profiles from several GUVs (n ≥ 13) were obtained following the procedure shown in Figure 3B. Error bars represent SEM. (B) Averaged intensity profile of Sec13/31 membrane binding with different lipid composition and conditions omitting Sar1 or with full COPII coats. Intensity profiles from several GUVs (n ≥ 10) were obtained following the procedure shown in Figure 3B. Error bars represent SEM.

Figure 5

Lysophospholipids Decrease Membrane Bending Rigidity and Facilitate COPII Recruitment to Exit Sites (A) Diagram of surface area of phosphatidylinositol 32:1 and surface area of lysophosphatidylinositol 16:0. (B) Deformable ER membrane with lysophospholipids facilitates the recruitment of COPII proteins. Insertion of Sar1 on the outer leaflet of the ER and subsequent recruitment of Sec23/24 could trigger the sorting of lysoPI toward disordered membrane (1). Enrichment in lysoPI lowers membrane rigidity and facilitates high curvature deformation by COPII proteins (2). Scission of vesicles is enriched in lysoPI (3).