Cargo crowding contributes to sorting stringency in COPII vesicles

Abstract

Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy contributes to ER retention: in the absence of abundant cargo, nonspecific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates a crowded lumen that drives selectivity. Retention of ER residents thus derives in part from the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier.

Figure 1.

3D ultrastructure of COPII budding events and free vesicles by CLEM. (A) Resin-embedded yeast cell containing ERES correlated to Sec24-sfGFP signal. Left panel, inset: two Sec24-sfGFP foci (green) are highlighted. Fiducial markers used for correlation are visible in green and magenta. Left panel, a virtual slice of a low magnification tomogram used for correlation with fluorescent image of the same cell (inset). Middle-left panel is a virtual slice of a high magnification tomogram on one of the two Sec24-sfGFP marked ERES, highlighted by a 500 x 500 nm square. Nuclear envelope and ER cisterna are false colored in yellow, and a free vesicle is highlighted in cyan. Middle-right panel is a zoom-in on the same virtual slice as the previous panel. The center of the 250-nm-diameter circle marks the predicted position of the GFP signal centroid. Colored arrowheads mark a nascent COPII bud (yellow) and a free vesicle (cyan), represented in a segmentation model on the farthest right panel. (B) A virtual tomographic slice showing a correlated Sec24-sfGFP ERES. Left panel, ER is false-colored in yellow. Central panel, arrowhead marks the emerging bud. Right panel is a segmentation model of the budding event. (C) Upper panels: two virtual tomographic slices of a single Sec16-sfGFP correlated spot: different z positions of the same x,y position are shown, revealing a multibudded ERES with two buds (yellow) and a free vesicle (cyan). Lower panels are a virtual tomographic slice showing a correlated Sec16-sfGFP ERES. Left panel, ER is false colored in yellow. Central panel, arrowheads mark an emerging bud (yellow) and a free vesicle (cyan). Right panel is a segmentation model of the budding event. Scale bars: A, EM, 500 nm; inset, 1 µm; all other panels, 100 nm.

Figure S1.

CLEM tomography of COPII-associated membranes. (A) Two virtual tomography slices of a single Sec24-sfGFP positive ERES. Different heights within the correlation are marked (z), showing a multibudded ERES with two buds (yellow) and a Golgi complex cisterna (purple). Scale bars, 100 nm. (B) Serial dilutions of the indicated strains were spotted as serial dilutions onto media containing 5-FOA to counter select for the SEC13-URA3 plasmid and test for viability. On standard media (left panel), all strains grew, whereas growth in the absence of SEC13 (5-FOA; right panels) was only observed in an emp24Δ background. Chromosomal tagging of SEC16 was tolerated in this background, whereas tagged SEC24 was not viable. (C) Fluorescence microscopy of the indicated strains expressing SEC16-sfGFP and a SEC24-sfGFP WT strain. Scale bars, 2 µm.

Figure 2.

Deletion of SEC13 results in pleomorphic membranes at ERES. (A) SEC16-sfGFP-positive ERES localized by CLEM in an emp24Δ sec13Δ cell. Upper and lower panels are different virtual slices from the same tomogram, representing different z-positions. Two buds form at the nuclear envelope (yellow) with six free vesicles (cyan) and two undefined tubular compartments (purple) in close proximity. In the central panels, colored arrowheads mark the same membrane structures. Right panels are two side views of a segmentation model of the ERES. (B) Table of ERES ultrastructure categories (percentages from total number [n] of correlated spots per yeast strain). (C) Plot of maximum diameter (nm) of vesicles for the different strains indicated; n = 15 for Sec24-sfGFP, n = 26 for Sec16-sfGFP, n = 35 for emp24Δ sec13Δ, n = 11 for emp24Δ. Bars correspond to median value and 95% confidence interval. Statistical test was a one-way ANOVA with Tukey’s correction for multiple comparisons; ns, not significant. (D) A SEC16-sfGFP-positive ERES localized by CLEM in an emp24Δ SEC16-sfGFP cell. A bud emerges from the nuclear envelope (yellow) with a free vesicle by the side (cyan). 3D segmentation model on the right. Scale bars, 100 nm.

Figure S2.

Virtual tomogram of an emp24Δ sec13Δ ERES. (A) Virtual tomography slices through the 3D volume (z) of an emp24Δ sec13Δ cell expressing SEC16-sfGFP. Left panels show colored ER (yellow), vesicles (cyan), and an unidentified tubular compartment (purple). Central panels show the same structures highlighted with colored arrowheads. Left panels are cut-throughs of a segmentation model of the 3D ultrastructure of membranes at the ERES. Scale bars, 100 nm.

Figure 3.

Kar2 secretion is not due to UPR, retrieval failure, or changes in ER lumenal mobility. (A) Serial dilutions of the indicated yeast strains were overlaid with nitrocellulose, and secreted Kar2 detected with Kar2-specific antibodies. (B and C) Kar2 was detected in intracellular (lysate) and secreted (media) fractions by SDS-PAGE and immunoblotting with anti-Kar2 antibodies. (D) Mobility of Kar2-sfGFP was measured by FLIP. Kar2 half-life in individual cells was measured in the indicated strains. WT + DTT cells were treated with 5 mM DTT for 1 h. The graph shows the mean and the error bars represent SD; n = 23 (WT); n = 30 (emp24Δ); n = 27 (WT + DTT). Statistical test was a one-way ANOVA with Dunnett’s correction for multiple comparisons. (E) Fluorescence microscopy of WT and emp24Δ cells expressing Erd2-GFP revealed ER localization in both strains.

Figure 4.

Elevated bulk flow stems from decreased cargo crowding. (A) WT and emp24Δ strains expressing GAL_pr-SP-FLAG-Cp were induced with 0.02% galactose, and separated into intracellular (lysate) and extracellular (media) fractions. Cp was detected by SDS-PAGE and anti-FLAG immunoblot. (B) FLAG-Cp was immunoprecipitated from media and lysate fractions of [³⁵S]methionine-labeled cells at the indicated times, analyzed by SDS-PAGE, and detected by autoradiography. The percentage of secreted FLAG-Cp is plotted for the indicated times. Error bars depict SD; n = 6. (C) As described in A. (D) As described in B. Error bars depict SD; n = 3. Statistical tests were t tests. (E) Gas1 and CPY maturation were examined in WT and ccw12Δ strains by pulse chase with [³⁵S]methionine. Gas1 and CPY were immunoprecipitated from lysates at the indicated times and detected by SDS-PAGE and autoradiography. (F) Fluorescence microscopy of WT and ccw12Δ cells expressing Emp24-sfGFP revealed similar localization of Emp24. (G) Steady-state levels of Erv25 in the indicated strains were measured from whole cell lysates by immunoblotting using an Erv25-specific antibody. A nonspecific band labeled with an asterisk is shown as loading control in A, C, and G.

Figure S3.

Loss of the abundant cell wall protein, Ccw12, phenocopies an emp24Δ strain. (A) Pie chart of GFP fluorescence of N-terminally tagged secretome proteins (Yofe et al., 2016). Cell wall proteins represent ∼25% of the GFP-secretome, with Ccw12 alone contributing ∼12% of the fluorescent signal. TA, tail-anchored; MP, membrane protein. (B) Serial dilutions of WT and ccw12Δ yeast strains were overlaid with nitrocellulose, and secreted Kar2 detected with Kar2-specific antibodies. (C) Mobility of Kar2-sfGFP was measured by FLIP. Half-time values, calculated as described in Fig. 3 D, of single cells are plotted for the indicated strains or WT-treated cells with 5 mM DTT for 1 h. Error bars represent SD; n = 23 (WT); n = 27 (WT + DTT); n = 23 (ccw12Δ). Statistical test was a one-way ANOVA with Dunnett’s correction for multiple comparisons. (D) Fluorescence microscopy of ccw12Δ cells expressing Erd2-GFP revealed ER localization.

Figure 5.

Restoring cargo occupancy reverses ER leakage. (A) Schematic of Emp24 and chimeras used in B; GOLD, Golgi dynamics domain; CC, coiled-coil; TM, transmembrane domain. (B) Kar2 secretion was analyzed in emp24Δ cells after galactose induction of the constructs indicated. Intracellular (lysate) and extracellular (media) proteins were resolved by SDS-PAGE and detected by Western blot against Kar2 and GFP.

Figure S4.

Characterization of modular Emp24 chimeras. (A) Fluorescence microscopy of emp24Δ cells expressing the indicated Emp24 chimeras. (B) Steady-state levels of Erv25 in WT cells and emp24Δ cells expressing the different chimeras indicated were measured from whole cell lysates by immunoblotting using an Erv25-specific antibody. Expression of chimeric proteins was detected from whole cell lysates using an antibody against GFP. A nonspecific band labeled with an asterisk is shown as loading control. (C) The amounts of secreted Kar2 and ER-GFP were analyzed in emp24Δ mutants with and without the ER-GFP plasmid. Secreted proteins in the extracellular media were concentrated using TCA; intracellular proteins were extracted with SDS. Intracellular (lysate) and extracellular (media) proteins were resolved by SDS-PAGE and detected by Western blot against Kar2 and GFP.

Figure 6.

LST1 deletion restores cargo stringency and reduces bulk flow. (A) Serial dilutions of the indicated strains were spotted onto YPD plates and Kar2 secretion determined by colony immunoblot as described in Fig. 3 A. (B) WT, lst1Δ, emp24Δ, and emp24Δ lst1Δ cells expressing HA-CPY* were subjected to pulse chase analysis. CPY* was immunoprecipitated from lysates at the indicated times and either analyzed directly or subjected to secondary immunoprecipitation using anti-α-1,6-mannose antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and detected by autoradiography. The ratio of Golgi-modified CPY* to total CPY* relative to a WT strain at t = 30 min was determined for three independent experiments. Averages and SDs are plotted (n = 3). (C) FLAG-Cp was detected in strains indicated after induction with 0.02% Galactose as described in Fig. 4 A (left panel). Pulse-labeled proteins were immunoprecipitated from the media and lysates at the indicated times as described in Fig. 4 B. Error bars depict SD; n = 3 (right panel); statistical test was a t test.

Figure 7.

The volume of COPII vesicles is significantly reduced in emp24Δ lst1Δ cells. (A) A Sec16-sfGFP–positive ERES in an emp24Δ lst1Δ cell. Left panel is a virtual tomographic slice showing a false-colored ER tube (yellow) and a free vesicle (cyan). Central panel shows the same structures highlighted by colored arrowheads. Right panel is a segmentation model of the corresponding 3D membrane ultrastructure showing a bud and two vesicles. Scale bar, 100 nm. (B) Plot of the volume (nm³) of COPII vesicles in the strains indicated. n = 26 for Sec16-sfGFP, n = 11 for emp24Δ, n = 46 for emp24Δ lst1Δ. Bars correspond to median value and 95% confidence interval. Statistical test was a one-way ANOVA with Tukey’s correction for multiple comparisons; ns, not significant. (C) Model of cargo crowding in vesicles. 2D sections of vesicles drawn to scale illustrating the cargo crowding effects in different strains. The radius of the vesicle lumen and the space occupied by the selected cargo layer (green) in a given vesicle determines the free volume available to bulk flow. Different bulk flow cargoes (cyan and purple circles) will access this space differently based on their size (see Materials and methods for calculation details). (D) Heatmap showing changes in K as a function of R_av and r_cargo. Depending on the relative size difference between the available size of vesicle and size of the cargo molecule, the partitioning process could either be less sensitive (relaxed partition) or more sensitive (stringent partition) to the size of a vesicle. Dashed lines mark the vesicle sizes associated with different genetic backgrounds. Solid lines mark the specific cargo, Cp (cyan) and a Kar2/client complex (purple). (E) Changes of K for Cp (cyan) and a Kar2/client complex (purple) as a function of vesicle size. Smaller vesicles tend to have more stringent partitioning compared with larger ones, and the size of vesicles impacts partitioning of larger bulk-flux cargoes more significantly than smaller cargoes (see Materials and methods for calculation details). Dashed lines mark the vesicle sizes associated with different genetic backgrounds.

Figure S5.

Partitioning model for cargo occupancy in a COPII vesicle. (A) Model of a COPII vesicle and parameters used in the calculation. The vesicle consists of a lipid bilayer (cyan), a cargo layer (pink), and available space (denoted by short dashed circle). Bulk flow cargoes of different sizes sample different effective volumes (denoted by long dashed circle) during diffusion as a result of the excluded volume effect (denoted by the yellow layer). The radius of effective volume (R_eff) is determined by the R_av and the r_cargo. (B) Parameters and results of calculation. K for smaller cargoes is greater than that of larger cargoes, but the percent change relative to WT is greater for the larger cargo. (C) Changes of K as a function of cargo size, dashed lines mark the size of Cp and a Kar2 complex discussed in this work. From the plot it is clear that smaller vesicles tend to have more stringent partition compared with larger ones.