COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers

Abstract

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER-ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins' role in ER-to-Golgi transport.

Figure 1.

Intracellular distribution of ERESs using FP-tagged COPII expressed in living intact cells. (A) Intracellular distribution of ERESs. A confocal image of a COS7 cell coexpressing the ER marker VSVG-YFP (left and green in merged image) at the nonpermissive temperature 39.5°C or the ER membrane marker reticulon-GFP (left insert and green in merged insert) and the COPII subunit Sec24C-mCherry (center, and red in merged image). Scale bars = 5 µm. (B) A HeLa cell clone expressing an endogenous (endo) Sec13-mCherry (red) inserted using the CRISPR/CAS12 knock-in method were transfected with VSVG-EGFP (green). Cells were transferred to permissive temperature (32°C) after an overnight incubation at 39.5°C, and images were taken at 15-s intervals for ∼40 min. See Video 1. Shown are images at 0 and 40 min. Scale bars = 5 µm.

Figure S1.

Characterization of HeLa cells with Sec13 endogenously tagged with mCherry. (A) Single apotome section of a pool of HeLa-L6 cells expressing endogenously tagged (endo) Sec13-mcherry costained with Sec31 antibodies. Sec13-mcherry nicely localizes to ERES. Scale bar = 5 µm. (B) Single apotome section of a pool of HeLa cells expressing Sec13-mcherry costained with Sec13. The untagged cells (asterisks) express similar levels of Sec13 as the Sec13-mcherry expressing cells, demonstrating that Sec13-mcherry is expressed at or below endogenous levels. Scale bar = 5 µm. (C) siRNA against Sec13, but not control (ctrl) siRNA, reduced expression of Sec13-mcherry as visualized by mCherry fluorescence and immunostaining against Sec13. Scale bars = 5 µm. (D) Western blot of HeLa and HeLa-L6 cells expressing Sec13-mcherry using Sec13 antibodies demonstrates efficiency of Sec13 siRNA. Sec13-mcherry is expressed below Sec13 levels and is efficiently reduced by Sec13 siRNA, validating that the identity of the Sec13-mcherry fusion.

Figure 2.

Membrane-associated COPII is stationary compared with cargo vesicle movement and microtubule plus-end polymerization. (A) Comparison between membrane-associated COPII movement and transport of ER-to-Golgi carriers. An inverted brightest pixel projection of 50 images from a 0.66-s interval time-lapse sequence of COS7 cell coexpressing VSVG-YFP (left, and green in merged image) and the COPII subunit Sec24C-mCherry (center, and red in merged image) after a shift to permissive temperature (32°C). See Video 2. Scale bar = 5 µm. (B) Comparison between membrane-associated COPII and microtubule plus-end polymerization using EB3-GFP. An inverted brightest pixel projection of 50 images from a 3.7-s interval time-lapse sequence of COS7 coexpressing EB3-GFP (left, and green in merged image) and the COPII subunit Sec24C-mCherry (center, and red in merged image). See Video 3. Scale bar = 10 µm.

Figure 3.

Analysis of cargo accumulation and carrier formation in living cells. (A) Accumulation of VSVG-YFP cargo and fission of carriers from ERESs in living cells. Confocal images were captured after the shift to the permissive temperature of COS7 cells coexpressing COPII subunit Sec24C-mCherry (red) and the cargo protein VSVG-YFP (green). See Video 4. Scale bar = 2 µm. (B) Cargo accumulation in a single ERES and carrier fission. Confocal images captured at 2.6-s intervals of COS7 cells transfected and treated as in A. VSVG-YFP (green) accumulation downstream to Sec24C-mCherry (red) is shown in two top panels and fission of carriers in the two bottom panels. See Video 5, Video 6, Video 7, and Video 8. Scale bar = 1 µm. (C) Analysis of ER export using the RUSH system. Images were captured after the addition of biotin to living COS7 cells 48 h after cotransfection of Sec24C-mCherry (red) and the cargo protein RUSH-TNF-GFP (green). Yellow arrowheads point to carrier elongating and budding. Scale bar = 1 µm. (D and E) Budding of a carrier from ERES in Sec13-mCherry (red) CRISPR/CAS12 knock-in HeLa cell clone transfected with VSVG-EGFP (green). White arrows and arrowheads point to carriers. Scale bar = 1 µm.

Figure 4.

Analysis of the life history of ER-to-Golgi carriers: budding translocation, fusion, and colocalization of cargo with the small GTPase Rab1b. (A) Photobleaching of the Golgi to expose ER-to-Golgi carrier life history. The Golgi region of interest was photobleached in the VSVG-GFP (green) channel after transfer to permissive temperature (32°C), following overnight at 39.5°C. Cells used were a HeLa cell clone expressing an endogenous (endo) Sec13-mCherry (center, and red in merged image) transfected with VSVG-GFP (bottom, and green in merged image). Scale bars = 5 µm. (B) Representative images from a time-lapse series captured after the FRAP in A. Arrowheads point to carrier translocating and fusing with the Golgi. Right: Projection of the images in B. See Video 9. Scale bar = 5 µm. (C) Rab1b localizes with cargo throughout the life history of ER-to-Golgi carriers. Huh7 cells were cotransfected with VSVG-YFP (center, and green in merged image) and Rab1-mCherry (red and inverted, bottom), were shifted to the permissive temperature of 32°C after an overnight in nonpermissive temperature (39.5°C). Representative images captured by confocal microscopy from a time-lapse series are shown with designated times. Arrowheads point to a single carrier budding from an ERES translocating and fusing with the Golgi apparatus. Right: A projection showing the path of the same carrier. See Video 10. Scale bar = 1 µm.

Figure 5.

Characterization and localization of COPII in ERESs in BFA/Noc-treated intact living cells. (A) 3D confocal analysis of ERESs in BFA/Noc-treated living cells. A single confocal image from a z-section stack of a COS7 cell cotransfected with VSVG-YFP (green) and Sec24C-mCherry (red) transferred to permissive temperature (32°C) after overnight incubation at 39.5°C and treated with BFA/Noc as described in Materials and methods. The top left insert is a fivefold enlargement of a single ERES. The top right insert is a 3D reconstruction of the same ERES. Bar and white arrows are 1 µm in the x, y, and z directions. Scale bar = 10 µm. (B) Colocalization of ERGIC53 and COPII in ERESs under BFA/Noc treatment. COS7 cells expressing ERGIC53-YFP and the COPII subunit Sec24C-mCherry were treated with BFA/Noc as described in Materials and methods section. Inset (scale bar = 2 µm) is a fivefold enlargement of a single representative ERES. Scale bar = 10 µm. (C) COPII-labeled membranes separate ER and ERES membranes. Confocal deconvolved images of a COS7 cell were transfected and treated as in A. Six representative fourfold enlarged images of ERESs are shown on the right. Yellow/blue arrows point to COPII-coated collars (Sec24C-mCherry, red) intersecting between ER and ERES. The VSVG (green) channel is shown inverted on the right. See Video 11 and Video 12. Scale bar = 10 µm. (D) ERESs are connected to the ER in BFA/Noc-treated cells. Confocal image of a living COS7 cell cotransfected with VSVG-YFP (green) and the soluble secreted luminal marker signal sequence-mCherry (red) under BFA/Noc treatment. See Video 13. Bar = 1 µm. (E) Immunogold EM analysis demonstrates the transformation of ERES membranes under BFA/Noc treatment to spherical-tubular membranes known as glumerolini. Left: Confocal image of an ERES of a fixed COS7 cell expressing Sec23-GFP (green) and VSVG-Scarlet (red) under BFA/Noc treatment after 30 min at the permissive temperature 32°C. Right: Immunogold EM serial sections labeled with anti-GFP antibodies of ERESs in BFA/Noc-treated cells. Yellow arrowheads point to sites of contact to ER with increased labeling of COPII. Scale bar = 200 nm. Bottom: A 3D reconstruction of the membrane structure from serial sections. Bottom right: A scheme depicting the positioning of the section relative to the ERESs under BFA/Noc treatment.

Figure S2.

Structure and function of ER exit sites in BFA/Noc treated cells. (A) Relative localization of Sec24C-mCherry (red) and VSVG (green) within ERES of BFA/Noc-treated COS7 cells. Top, left to right: (1) Confocal microscope. (2) Immunofluorescence analysis of endogenous Sec24C (red) in a cell expressing VSVG-YFP (green). (3) Cells under Golgicide A/Colchicin treatment. (4) Deconvolution using DeltaVision microscope. Bottom, left to right: (1) Multifocal structured illumination microscopy (M-SIM). (2) Relative localization of Sec24C-mCherry (red) and ERGIC53-YFP, GalT-YFP, or CFTR-GFP (green) within a representative ERES of COS7 cells under BFA/Noc treatment. Scale bars = 1 µm. (B) Cargo and membrane accumulation in a single dilated exit site. A COS7 cell transfected with cargo protein VSVG-YFP and treated with BFA/Noc was monitored by a spinning disk confocal microscope captured at 5 frames/s. Representative time-lapse images of a single dilated exit site marked by the white square frame, are shown in the middle panel. Bottom: A kymogram generated from the line marked by the yellow line. Scale bar = 1 µm. (C) Analysis of COPII-mediated cargo sorting dynamics in ERESs of BFA/Noc-treated living COS7 cells. A cell coexpressing VSVG-YFP (green) and Sec24C-mCherry (red) was imaged using a spinning disk confocal microscope at 0.2-s intervals at the permissive temperature 32°C. The fluorescence intensity of VSVG-YFP (green line) and Sec24C-mCherry (red line) in three representative ERESs I through III (in white frames) are plotted, and representative images are shown on the right. Black arrowheads in the graph show time points of images on the right. Scale bar = 5 µm. (D) The mutant cargo protein CFTRΔ508-GFP (green, bottom) is excluded from ERES in BFA/Noc-treated COS7 cells. The cells are coexpressing Sec24C-mCherry (red) with either WT CFTR-GFP (green, top) or CFTRΔ508-GFP (green, bottom). Insets are enlarged fivefold. Left scale bars = 10 µm. Right scale bars = 1 µm.

Figure 6.

A site-directed gain of function mutation of the cargo binding site of Sec24B obstructs ER export at the ER–ERES boundary. (A) Computer modeling of the binding of Sec24B WT or the V932R mutant to the VSVG export motif. (A) A ribbon diagram of the crystallographic structure of the binding pocket of Sec24B is shown in gray with allowed conformations of the VSVG export motif in colors. In green are the amino acids with side chain structural representations before and after substitution. (B) Electrostatic potential and solvent accessibility maps demonstrate a more defined pocket for the VSVG tail binding site in the V932R mutant compared with the WT. (C) FRAP of COS7 cells coexpressing the VSVG-GFP (green) and the Sec24B-mCherry (red, WT on the left and V932R mutant on the right) under BFA/Noc treatment as described in Materials and methods. Images were captured after bleaching a rectangle of WT or mutant Sec24B-mCherry to follow the recovery rate. Scale bar = 5 µm. (D) FRAP analysis for the experiments in C. Comparison of the membrane-binding dynamics for the WT (black) or V932R mutant (red) Sec24B. To avoid background fluorescence, maximum pixel intensities within the bleach box were exclusively plotted. Data represent an average of 10–12 experiments. Data were fitted to a single exponential equation shown in the graph. Bar graphs show the average mobile fractions (left) and exponential rate constant (right) with SDs. (E) Dose–response of Sec24B_V932R mutant (red) overexpression inhibiting ER-to-Golgi export of VSVG-YFP (green). Three cells with different levels of expression of Sec24B_V932R with a corresponding degree of ER export inhibition. Arrowhead points to the Golgi apparatus. Scale bar = 10 µm. (F) Expression of Sec24B_V932R causes VSVG retention in the ER. Shifting cells coexpressing the mutant Sec24B_V932R (red) and VSVG-YFP (green) to permissive temperature results in retention of VSVG-YFP in ER (blue arrow). Yellow arrowhead points to the Golgi apparatus in a cell not expressing Sec24B_V932R. Scale bar = 10 µm. (G) Sec24B_V932R  prevents VSVG cargo accumulation in ERESs in BFA/Noc-treated cells. COS7 cells were cotransfected with VSVG-YFP (geen) and Sec24B_V923R-mCherry (red) and treated with Noc and BFA. Bottom left cell is expressing the Sec24B_V932R-mCherry mutant where VSVG-YFP is retained in the ER. Scale bar = 10 µm. (H) Sec24B_V932R blocks the accumulation of CFTR in ERESs in BFA/Noc-treated cells. COS7 cells cotransfected with CFTR-GFP (green, inverted on the right) and either Sec24B_V923R-mCherry or Sec24B_WT-mCherry (red) were treated with Noc and BFA. Yellow arrowheads point to ERESs. Scale bars = 10 µm.

Figure S3.

The Sec24B_V932R mutant interacts with VSVG. Immunoprecipitation (IP) analysis demonstrating interaction between the Sec24B_V932R mutant and VSVG. Cells coexpressing VSVG-YFP and WT or mutant Sec24B were immunoprecipitated with anti-VSVG antibodies, separated on SDS-PAGE, blotted, and probed with anti-GFP and anti-mCherry for VSVG and Sec24B, respectively. WB, Western blot.

Figure 7.

Schematic representation for the localization and function of the COPII heterocomplex at the ER–ERES boundary: Support for direct ERES to Golgi transport. (A) The localization and function of the COPII heterocomplex at the ER–ERES boundary. Main panel: COPII dynamically binds and establishes domains of collar-like elongated membranes that comprise a stable ER–ERES boundary. Cargo accumulates in COPII-free ERES membranes by passage through the COPII coated neck. Fission ensues to form Golgi bound COPII-deficient carriers. Top right: Hydrophobic mismatching between transmembrane domains of cargo proteins and surrounding lipids is essential for cargo sorting: Schematic representation demonstrating how bilayer thickness gradient from thinner ER to thicker ERES membranes drives cargo into ERES. Alleviation of hydrophobic mismatching of cargo transmembrane domains facilitates their concentration in ERES membranes by preventing their diffusion through the COPII neck back to the ER. Bottom right: A detailed model of the COPII cargo sorting machinery at the ER–ERES boundary. COPII is recruited exclusively at the ER adjacent to the COPII neck by Sec12 and Sar1-GTP and binds cargo via the Sec24 subunit. The COPII-cargo complex is driven toward ERES potentially by the abovementioned hydrophobic mismatch interactions. COPII coat disassembly initiated by Sar1-GTP hydrolysis releases the cargo at the distal ERES end of the COPII-coated neck. (B) Golgi-associated ERESs in living intact cells. The intracellular distribution of COPII subunit Sec24C-mCherry (red) and Rab1b-YFP were used here as a Golgi marker. A confocal image of the Huh7 cell coexpressing the Rab1b-YFP (left, and green in merged image) and the COPII subunit Sec24C-mCherry (center, and red in merged image). Golgi apparatus marked by G. The frame showing the Golgi apparatus is enlarged threefold below. Scale bar = 5 µm. (C) Stable Golgi-associated ERES during cargo accumulation in Golgi apparatus. Time-lapse analysis of living cells coexpressing Sec24C-mCherry (red) and VSVG-YFP (green), showing an accumulation of cargo VSVG-YFP in Golgi membranes decorated with COPII-labeled domains. The area in white rectangles is magnified 3.3-fold on the bottom. Scale bars in top and bottom panels are 10 and 5 µm, respectively. (D) Golgi-associated ERESs disappear upon BFA-induced Golgi membranes blink-out. Representative images from a time-lapse sequence taken at 3-s intervals after addition of 5 µg/ml BFA to living cells coexpressing GalT-YFP (green, bottom) and Sec24C-mCherry (red and inverted, middle). Times are counted. Scale bar = 5 µm.

Figure S4.

Colocalization to and translocation from ERESs of COPI-coated membranes. Time-lapse microscopy analysis of living LDLF cells stably expressing ε-COP-YFP (green) cotransfected with Sec24C-mCherry (red) and KDEL-BFP (Blue). Yellow arrows point to ERES labeled with Sec24C, and yellow arrowheads point to COPI membranes moving toward the Golgi (G). Scale bar = 10 µm.