Journal: Journal of cell science

Article Title: The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic assembly of SNARE complexes.

doi: 10.1242/jcs.122101

Figure Lengend Snippet: Fig. 1. Identifying interactions between the COG subunits and a subset of Golgi SNARE and SM proteins. The interactions between the eight COG subunits and components of the two Golgi SNARE complexes, the Stx5 and Stx6 SNARE complexes and their corresponding SM proteins, Sly1 and Vps45, respectively, were determined by co-IP experiments. The eight COG subunits were expressed as Myc-tagged proteins in HEK293 cells and their interaction with the indicated endogenous SNARE and SM proteins was determined by IP with anti-Myc antibody and immunoblotting (IB) with the indicated anti-SNARE or anti-SM protein antibody. The expression level of the endogenous SNARE and SM proteins was determined by IB of 10% of total cell lysates with the corresponding antibodies (Total; right panels).

Article Snippet: Polyclonal anti-Vti1a and anti-Vps45 antibodies were purchased from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Expressing

Journal: Journal of cell science

Article Title: The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic assembly of SNARE complexes.

doi: 10.1242/jcs.122101

Figure Lengend Snippet: Fig. 2. The Cog4 subunit interacts directly with the tSNAREs Vti1a, Stx16 and GS28 and the SM protein Vps45. (A,B) Cog4 interacts directly with Vti1a (A) and with Stx16 (B). The full-length Cog4 subunit and the cytosolic portion of either Vti1a or Stx16 or Stx6 were separately expressed in bacteria as His-tagged proteins and purified on Talon beads. Purified recombinant Cog4 and either Vti1a or Stx16 or Stx6 proteins were mixed together, incubated overnight and their interaction was determined by IP with anti-Cog4 antibody followed by IB with the indicated antibodies. 10% of the recombinant proteins used in the assays (Input) are shown on the left lanes. (C,D) Cog4 interacts directly with GS28 and Vps45. The cytosolic portion of GS28 or Stx6 (C) or the full-length Vps45 (D) was expressed in bacteria as GST-fusion protein, immobilized on glutathione-agarose beads and incubated with recombinant His-tagged full-length (F.L) Cog4 (C,D) or its truncated mutants (D). The interaction between the indicated recombinant proteins was determined by glutathione-beads pull-down experiments followed by IB with anti-His antibody. 10% of the recombinant proteins used in the assays (Input) are shown on the left lanes. (E,F) The SNARE domains of Vti1a (E) and Stx16 (F) interact with Cog4. The indicated GST-fusion proteins were purified from bacteria and immobilized on glutathione-agarose beads. The glutathione beads (GB) were then incubated with cell lysates of HEK293 cells expressing either Myc-Cog4 (E,F), Myc- Vps45 (F) or HA-Stx6 (E) as indicated, and their interactions were assessed by IB with either anti-Myc (E,F) or anti-HA antibody (E) antibody. Input: 10% of HEK293 cell lysates. The domain organization of Vti1a (E) and Stx16 (F) are shown in the schemes. The positions of the three-helical bundle (Habc), the SNARE motif (SNARE) and the trans-membrane domain (TM) are indicated in amino acid residues. For Stx16, the position of the N-terminal peptide (N-ter), which binds Vps45, is also indicated. (G) The N-terminal fragment of Cog4 (aa 1–231) interacts with Stx16. The cytosolic portion of Stx16 was expressed as GST-fusion protein in bacteria, immobilized on glutathione- agarose beads, and incubated with recombinant His-tagged Cog4 or its truncated mutants. The interactions between Stx16 and full-length Cog4 or its mutants were determined by IB with anti-His antibody. In all the glutathione-agarose beads pull-down experiments (C–G), GST was used as control. The expression level of GST and GST-fusion proteins was determined by Coomassie blue staining and is shown in the right panels (C–G).

Article Snippet: Polyclonal anti-Vti1a and anti-Vps45 antibodies were purchased from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Bacteria, Purification, Recombinant, Incubation, Expressing, Membrane, Control, Staining

Journal: Journal of cell science

Article Title: The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic assembly of SNARE complexes.

doi: 10.1242/jcs.122101

Figure Lengend Snippet: Fig. 3. Cog4 depletion affects the subcellular distribution of Stx6, Stx16, Vti1a and VAMP4 and their assembly into the SNARE complex. (A) HeLa cells were transfected with control or Cog4 shRNA construct. At 24 hours post transfection, cells were incubated with 1 mg/ml puromycin for 72 hours. Subsequently, total cell lysates were prepared and the downregulation of Cog4 was determined by IB with anti-Cog4 antibodies. The mean values6s.d. of three independent experiments are shown. (B) The steady-state level of Stx6, Stx16, Vti1a and VAMP4 was determined by western blotting of total cell lysates of control (pSuper- puro), Cog6-depleted (KD) and Cog4-depleted HeLa cells using the indicated antibodies. Densitometry analysis of the western blot signals was used to estimate the relative level of each SNARE in the Cog6- and Cog4-depleted cells as compared with the control HeLa cells. The mean values6s.d. of three independent experiments are shown in the table. (C) The subcellular distribution of Stx6, Stx16, Vti1a, VAMP4 and their associated SM protein Vps45 was determined by cell fractionation followed by western blotting using the indicated antibodies. The distribution of each SNARE between the cytosol (Cyt.), heavy membrane (HM) and light membrane (LM) fractions in the control and Cog4-depleted HeLa cells is shown at the upper panels. The relative distribution of each SNARE between the heavy and light membrane fractions was calculated for three independent experiments, and mean values6s.d. are shown at the bottom. Cells in B and C were transfected with the corresponding shRNA constructs and 24 hours post transfection treated with 1 mg/ml puromycin for 72 hours to select for homogenous population expressing the shRNAs constructs. (D) HeLa cells were transiently transfected with a Cog4 shRNA construct, and 72 hours later the cells were fixed and double immunostained with the indicated antibodies. The localization of the indicated SNAREs to the TGN or the Golgi membranes was determined by co- localization with Golgin97 or p115, respectively. As seen, the Golgi morphology of Cog4-depleted cells (marked by arrowheads) is strikingly different from the control cells. The four SNAREs could be partially detected on the Golgi-derived structures, and mainly appeared as small punctate cytosolic structures and diffuse cytosolic haze. Scale bars: 10 mm. (E) SNARE complex assembly is impaired in Cog4-depleted cells. Control (pSuper puro) and Cog4-depleted HeLa stable cell- lines (KD 1&2) were pretreated with NEM, solubilized and subjected to IP with either anti-Vti1a or anti-VAMP4 antibody. The presence of the indicated SNARE proteins in the immunocomplexes was determined by IB using the corresponding antibodies.

Article Snippet: Polyclonal anti-Vti1a and anti-Vps45 antibodies were purchased from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Transfection, Control, shRNA, Construct, Incubation, Western Blot, Cell Fractionation, Membrane, Expressing, Derivative Assay, Stable Transfection

Journal: Journal of cell science

Article Title: The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic assembly of SNARE complexes.

doi: 10.1242/jcs.122101

Figure Lengend Snippet: Fig. 5. Cog4 enhances the assembly of the Stx6 SNARE complex. (A,B) Cog4 facilitates the assembly of Stx6-Stx16-Vti1a tSNARE complex in vitro. The cytosolic portion of Stx16 (A) or Vti1a (B) was expressed in bacteria as GST-fusion protein, immobilized on glutathione-agarose beads and incubated with cell lysates of HEK293 cells expressing the indicated proteins. The interactions between the indicated SNAREs were determined by glutathione-agarose beads pull- down experiment followed by IB with anti-HA antibody. 10% of HEK293 lysate (Input) was used for assessing the expression levels of the indicated proteins. GST was used as control in the pull-down experiments, and its expression level as well as the level of the other GST-fusion proteins was determined by Coomassie blue staining and is shown on the right panels. (C) Cog4 enhances the interactions between Stx16, Vti1a and Stx6 in intact cells. Myc-Stx16 and HA-Vti1a (left panel) or Myc-Stx16, HA-Vti1a and HA-Stx6 (right panel) were co-expressed in HEK293 cells either alone or together with HA-Vps45 or HA-Cog4. The interaction between Stx16 and its SNARE partners was determined by IP with anti-Myc antibody and IB with anti-HA antibody. The expression levels of HA- Vti1a and HA-Stx6 are shown on the lower panels. (D) Cog4 enhances the interaction of VAMP4 with its cognate tSNAREs in vitro. The cytosolic portion of Stx16 was expressed in bacteria as GST fusion protein, immobilized on glutathione-agarose beads and incubated with purified recombinant His-tagged Vti1a, Stx6 and VAMP4 in equal molar ratios, either alone or in the presence of recombinant His-tagged Cog4. The interaction between VAMP4 and its cognate tSNAREs was determined by glutathione-agarose beads pull-down experiment followed by IB with anti-VAMP4 antibody. GST was used as control.

Article Snippet: Polyclonal anti-Vti1a and anti-Vps45 antibodies were purchased from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: In Vitro, Bacteria, Incubation, Expressing, Control, Staining, Purification, Recombinant

Journal: Journal of cell science

Article Title: The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic assembly of SNARE complexes.

doi: 10.1242/jcs.122101

Figure Lengend Snippet: Fig. 6. The interplay between Cog4 and two distinct Golgi SNARE complexes. (A,B) Cog4 inhibits nonfusogenic interactions between the Golgi tSNAREs Stx5 and Stx6. Control (pSuper puro) and Cog4-depleted HeLa stable cell-lines (KD) were pretreated with NEM, solubilized and subjected to IP with anti-Stx5 (A) or anti-Stx6 (B) antibody. The presence of the indicated SNARE proteins in the isolated immunocomplexes was determined by IB with the corresponding antibodies. Whereas the interaction of Stx5 with its cognate tSNARE (GS28) and vSNARE (GS15) is reduced in Cog4-depleted cells, its interaction with Stx6 is increased. Two different Cog4-depleted HeLa stable cell-lines were used for the analysis. Stx5 appears as a double band corresponding to its two isoforms. (C–E) The interaction of Cog4 with one SNARE complex can inhibit its interaction with the second complex. (C) Stx16 competes with Stx5 for Cog4 binding. Recombinant His-tagged Cog4 (0.2 mg) was pre-incubated with increasing concentrations of recombinant His-tagged Stx16 at the indicated molar ratios. The protein mixtures were then incubated with GST-Stx5 immobilized on glutathione-agarose beads. The interaction between Cog4 and GST-Stx5 was determined by glutathione-agarose beads pull-down experiment followed by IB with anti-His antibody. (D) The SM protein Vps45 competes with Sly1 for Cog4 binding. Recombinant His-tagged Cog4 (0.2 mg) was pre-incubated with increasing concentrations of recombinant His-tagged Vps45 at the indicated molar ratios. The protein mixtures were then incubated with GST-Sly1 immobilized on glutathione-agarose beads. The interaction between Cog4 and GST-Sly1 was determined by glutathione-agarose beads pull-down experiment followed by IB with anti-His antibody. 10% of the protein mixtures (Input) were used for assessing the amounts of the purified proteins. GST was used as control. (E) Overexpressed HA-Vti1a competes with endogenous Stx5 for Cog4 binding. HEK293 cells were transfected with a constant amount of the expression vector encoding for Myc-Cog4 and increasing amounts of the expression vector encoding for HA-Vti1a. The interaction between Myc-Cog4 and endogenous Stx5, in the presence of increasing amounts of HA-Vti1a, was determined by IP with anti-Myc antibodies followed by IB with the indicated antibodies. The expression levels of endogenous Stx5 are shown in the lower panel.

Article Snippet: Polyclonal anti-Vti1a and anti-Vps45 antibodies were purchased from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Control, Stable Transfection, Isolation, Binding Assay, Recombinant, Incubation, Purification, Transfection, Expressing, Plasmid Preparation