TFG clusters COPII-coated transport carriers and promotes early secretory pathway organization

Abstract

In mammalian cells, cargo-laden secretory vesicles leave the endoplasmic reticulum (ER) en route to ER-Golgi intermediate compartments (ERGIC) in a manner dependent on the COPII coat complex. We report here that COPII-coated transport carriers traverse a submicron, TFG (Trk-fused gene)-enriched zone at the ER/ERGIC interface. The architecture of TFG complexes as determined by three-dimensional electron microscopy reveals the formation of flexible, octameric cup-like structures, which are able to self-associate to generate larger polymers in vitro. In cells, loss of TFG function dramatically slows protein export from the ER and results in the accumulation of COPII-coated carriers throughout the cytoplasm. Additionally, the tight association between ER and ERGIC membranes is lost in the absence of TFG. We propose that TFG functions at the ER/ERGIC interface to locally concentrate COPII-coated transport carriers and link exit sites on the ER to ERGIC membranes. Our findings provide a new mechanism by which COPII-coated carriers are retained near their site of formation to facilitate rapid fusion with neighboring ERGIC membranes upon uncoating, thereby promoting interorganellar cargo transport.

Keywords: COPII vesicle transport; intrinsic disorder; secretion; single particle electron microscopy.

Figure 1

TFG octamers assemble into a cup-like structure in vitro

1. Montage of class averages and 3D volumes of human TFG (amino acids 1-193; EMDataBank accession code EMD-6076). Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference-free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left. Scale bars, 50 Å.

2. Montage of class averages and 3D volumes of C. elegans TFG (amino acids 1-195; EMDataBank accession code EMD-6075), generated as described in (A). Scale bars, 50 Å.

3. Superposition of 3D volumes of full-length human TFG and truncated TFG (amino acids 1-193). The truncated form is depicted in yellow, while the full-length form is depicted as gray mesh. The top view (top) exhibits limited differences in the structures, while the side view (bottom) shows extra density in the full-length TFG isoform. Scale bar, 25 Å.

Figure 2

The PQ-rich domain of TFG facilitates its further polymerization

1. TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations. Error bars represent mean ± SEM; n = 10 replicates.

2. Confocal images of recombinant, BODIPY-labeled TFG in the presence of varying potassium acetate concentrations. Scale bar, 2 μm.

3. Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain-EM. Large arrowheads highlight TFG polymers (only found in the presence of elevated potassium acetate). Small arrowheads highlight individual 11 nm cup-like TFG octamers. Scale bar, 30 nm.

4. Circular dichroism spectroscopy was used to characterize the carboxyl-terminus of C. elegans TFG (amino acids 195-486). Samples were analyzed at different concentrations, and the data were normalized relative to one another. CD spectra were collected at 25°C in 25 mM sodium phosphate (pH 7.2) using a 1 mm path length quartz cell. The spectra are characteristic of an intrinsically disordered protein.

Figure 3

TFG accumulates at the ER/ERGIC interface in mammalian cells

1. The distributions of endogenous TFG and ERGIC-53 were examined in hTERT-immortalized RPE-1 cells stably expressing low levels of mApple-Sec16B using SR-SIM (more than 15 cells analyzed on 3 separate occasions). Scale bar, 5 μm. Inset scale bar, 1 μm.

2. Cells stably expressing low levels of GFP-Sec16B and mRuby-Sec23A (Sec23A) were fixed and imaged using SR-SIM (n = 12). Scale bar, 5 μm. Inset scale bar, 1 μm.

3. The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby-Sec23A (Sec23A) using SR-SIM (n = 10). Scale bar, 5 μm. Inset scale bar, 1 μm.

4. The distributions of endogenous TFG and Sec31A were examined using g-STED (n = 8). Scale bar, 1 μm.

5. The distributions of endogenous TFG and Sec31A were examined using immunogold EM. Large arrowheads highlight 15 nm gold particles bound to α-TFG antibodies. The small arrowhead highlights 5 nm gold particles bound to α-Sec31A antibodies. At least 15 different cells were examined, and representative images are shown. Scale bar, 100 nm.

Figure 4

The PQ-rich domain of TFG is required for its distribution at the ER/ERGIC interface

1. A Cells depleted of endogenous TFG were transfected with a variety of TFG transgenes (including full-length, untagged TFG, a truncation mutant encoding amino acids 1-300 of untagged TFG, and a truncation mutant encoding amino acids 1-350 of untagged TFG) and stained using α-TFG and α-Sec31A antibodies. Scale bar, 5 μm. Inset scale bar, 1 μm. Images shown are representative of at least 15 individual cells analyzed for each condition.

2. B A recombinant polyhistidine- and SUMO-tagged TFG fragment (Sumo-TFG^c, amino acids 194-400; total molecular mass of 42 kDa) was expressed and purified from Escherichia coli extracts using nickel affinity resin. Coomassie-stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column (top) or a 10–30% glycerol gradient (bottom) are shown. To determine the native molecular weight of the protein, the following equation was used: M = 6πηNas/(1 − υρ), where M is the native molecular weight, η is the viscosity of the medium, N is Avogadro's number, a is the Stokes radius, s is the sedimentation value, υ is the partial specific volume, and ρ is the density of the medium (Siegel & Monty, 1966). These data suggest that this fragment of TFG is capable of forming dimers in solution.

3. C, D Control cells (C) or cells depleted of endogenous TFG (D) were transfected with a construct expressing TFG with a carboxyl-terminal GFP tag and stained using α-Sec31A antibodies. Scale bar, 5 μm. Images shown are representative of at least 20 individual cells analyzed for each condition.

Figure 5

TFG depletion disrupts early secretory pathway organization

1. Human RPE-1 cells were mock-transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α-Sec16A and α-Sec31 antibodies and imaged using confocal microscopy. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and Sec31A in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Inset scale bar, 1 μm.

2. Quantification of the number of Sec31A-labeled structures in a 4 μm² region. For both conditions (control or TFG siRNA-treated), at least 50 distinct regions away from the peri-nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P < 0.01 compared with control, calculated using a paired t-test.

3. Quantification of the number of Sec16A-labeled structures in a 4 μm² region. For both conditions (control or TFG siRNA-treated), at least 50 distinct regions away from the peri-nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. No statistically significant difference was observed, following analysis using a paired t-test.

4. Cells stably expressing low levels of GFP-ERGIC-53 and mRuby-Sec23A were fixed and imaged using SR-SIM (top), or depleted of endogenous TFG prior to fixation and SR-SIM imaging (bottom). Images shown are representative of at least 15 individual cells analyzed for each condition. Scale bar, 5 μm. Inset scale bar, 1 μm.

5. Human RPE-1 cells were mock-transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α-Sec16A and α-ERGIC-53 antibodies and imaged using SR-SIM. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and ERGIC-53 in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Arrowheads highlight Sec16A-labeled structures that do not exhibit juxtaposed ERGIC-53 staining. Inset scale bar, 1 μm.

6. Based on structured illumination microscopy, quantification of the number of Sec16A-labeled structures juxtaposed (within 500 nm) to ERGIC-53-labeled structures is shown. For both conditions (control or TFG siRNA-treated), at least 1,000 unique Sec16A-labeled structures were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P < 0.01 compared with control, calculated using a paired t-test.

Figure 6

TFG depletion delays de novo Golgi assembly

1. Human RPE-1 cells stably expressing low levels of mannosidase II-mApple (ManII-mApple) were mock-transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and ManII in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.

2. Human RPE-1 cells were mock-transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and GM130 in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.

3. Higher magnification views of the indicated regions in (B, boxed) are shown (GM130 distribution only). Scale bar, 2 μm.

Figure 7

TFG depletion causes the accumulation of cargo-laden COPII transport carriers throughout cells

1. Human RPE-1 cells stably expressing low levels of mannosidase II-mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with brefeldin A (BFA) for 1 h, followed by a wash into fresh media and further incubation for 2 h in the absence of BFA, prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A in green and ManII in red are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.

2. Human RPE-1 cells stably expressing low levels of mannosidase II-mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with BFA for 1 h, followed by a wash into fresh media and further incubation for 1 h in the absence of BFA, prior to fixation and staining using α-Sec16A and α-Sec31A antibodies. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A (green), ManII (red), and Sec16A (blue) are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.

3. Higher magnification views of the indicated regions in (B, boxed) are shown. Arrows highlight COPII-positive transport carriers that contain the cargo ManII, which are not juxtaposed to Sec16A-labeled sites on the ER. Additionally, arrowheads point out distinct foci in which COPII continues to associate with Sec16A-labeled sites, indicating that COPII vesicle formation continues in the absence of TFG. Scale bar, 1 μm.

Figure 8

Model for TFG function at the ER/ERGIC interface A model depicting the assembly of TFG at the ER/ERGIC interface, which facilitates the transient, local retention of COPII vesicles prior to vesicle uncoating and maintains the juxtaposed organization of ER and ERGIC membranes.