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Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells.

Zolov SN, Lupashin VV - J. Cell Biol. (2005)

Bottom Line: In this work we used short interfering RNA strategy to achieve an efficient knockdown (KD) of Cog3p in HeLa cells.Fragmented Golgi membranes maintained their juxtanuclear localization, cisternal organization and are competent for the anterograde trafficking of vesicular stomatitis virus G protein to the plasma membrane.In a contrast, Cog3p KD resulted in inhibition of retrograde trafficking of the Shiga toxin.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

ABSTRACT
The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved multi-subunit protein complex that regulates membrane trafficking in eukaryotic cells. In this work we used short interfering RNA strategy to achieve an efficient knockdown (KD) of Cog3p in HeLa cells. For the first time, we have demonstrated that Cog3p depletion is accompanied by reduction in Cog1, 2, and 4 protein levels and by accumulation of COG complex-dependent (CCD) vesicles carrying v-SNAREs GS15 and GS28 and cis-Golgi glycoprotein GPP130. Some of these CCD vesicles appeared to be vesicular coat complex I (COPI) coated. A prolonged block in CCD vesicles tethering is accompanied by extensive fragmentation of the Golgi ribbon. Fragmented Golgi membranes maintained their juxtanuclear localization, cisternal organization and are competent for the anterograde trafficking of vesicular stomatitis virus G protein to the plasma membrane. In a contrast, Cog3p KD resulted in inhibition of retrograde trafficking of the Shiga toxin. Furthermore, the mammalian COG complex physically interacts with GS28 and COPI and specifically binds to isolated CCD vesicles.

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The COG complex interacts with retrograde Golgi SNARE GS28 and β-COPI. (A and B) Protein complexes from detergent-solubilized rat liver Golgi were IP using anti-Cog3p (A, lane 1; B, lane 2), anti-GS28 (A, lane 4), preimmune IgGs (A, lane 3), or anti-PDI (B, lane 3). 10% of the Golgi lysates were loaded as a control (A, lane 2; B, lane 1). Note that Cog3p, GS28, and β-COPI were not recovered with control beads or beads loaded with anti-PDI antibodies (A, lane 3; B, lane 3). (C) CCD vesicles are partially COPI coated and do not carry VSVG. COG3 KD cells that express VSVG-GFP were fixed and processed for IF with mouse anti-GS15 antibodies and rabbit anti–ɛ-COPI antibodies as described in Materials and methods. All images were acquired with 63× objective and deconvolved. Note that a number of GS15-labeled CCD vesicles were colabeled with antibodies to ɛ-COPI coat (inset, arrowheads). VSVG was partially colocalized with the GS15 only on a fragmented Golgi membrane (inset, membranes labeled with asterisk), but not on CCD vesicles. Bar, 10 μm. (D) CCD vesicles do not significantly colocalize with the ER. COG3 KD cells were fixed and stained with rabbit anti-Sec61p (red) and mouse anti-GS15 IgGs (green). Bar, 10 μm. (E) CCD vesicles are distinct from early endosomes. COG3 KD cells were fixed and stained with rabbit IgGs to GPP130 (red) and mouse anti-EEA1 IgGs (green). Bar, 10 μm.
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fig9: The COG complex interacts with retrograde Golgi SNARE GS28 and β-COPI. (A and B) Protein complexes from detergent-solubilized rat liver Golgi were IP using anti-Cog3p (A, lane 1; B, lane 2), anti-GS28 (A, lane 4), preimmune IgGs (A, lane 3), or anti-PDI (B, lane 3). 10% of the Golgi lysates were loaded as a control (A, lane 2; B, lane 1). Note that Cog3p, GS28, and β-COPI were not recovered with control beads or beads loaded with anti-PDI antibodies (A, lane 3; B, lane 3). (C) CCD vesicles are partially COPI coated and do not carry VSVG. COG3 KD cells that express VSVG-GFP were fixed and processed for IF with mouse anti-GS15 antibodies and rabbit anti–ɛ-COPI antibodies as described in Materials and methods. All images were acquired with 63× objective and deconvolved. Note that a number of GS15-labeled CCD vesicles were colabeled with antibodies to ɛ-COPI coat (inset, arrowheads). VSVG was partially colocalized with the GS15 only on a fragmented Golgi membrane (inset, membranes labeled with asterisk), but not on CCD vesicles. Bar, 10 μm. (D) CCD vesicles do not significantly colocalize with the ER. COG3 KD cells were fixed and stained with rabbit anti-Sec61p (red) and mouse anti-GS15 IgGs (green). Bar, 10 μm. (E) CCD vesicles are distinct from early endosomes. COG3 KD cells were fixed and stained with rabbit IgGs to GPP130 (red) and mouse anti-EEA1 IgGs (green). Bar, 10 μm.

Mentions: We have previously demonstrated that the yeast COG complex regulates intra-Golgi retrograde trafficking and interacts with the essential components of core machinery of vesicular Golgi traffic, i.e., intra-Golgi SNARE proteins and COPI vesicular coat (Suvorova et al., 2002). To test if the mammalian COG complex may also interact with both SNARE and COPI components, we isolated Golgi from the rat liver and performed an IP of the COG complex, v-SNARE GS28, and PDI from detergent-solubilized membranes. WB analysis of coIP proteins revealed that both GS28 (∼5% from total) and small amounts of t-SNARE Syntaxin5 were coprecipitated with the Cog3p (Fig. 9 A, lane 1; unpublished data). It was previously shown that Syntaxin5 and GS28 form a Golgi-localized SNARE complex (Hay et al., 1997) and our findings suggest that the COG complex can interact with either individual SNARE proteins or with the entire SNARE core complex. In the reciprocal precipitation, Cog3p was coIP by anti-GS28 antibodies (Fig. 9 A, lane 4). Neither Cog3p nor the Golgi SNARE molecules were precipitated with control beads (Fig. 9 A, lane 3). We have also determined that the β subunit of the COPI coat was specifically precipitated with anti-Cog3p antibodies (Fig. 9 B, lane 2). These results indicated that the mammalian COG complex like its yeast homologue could physically interact with both vesicular and Golgi-localized components of membrane transport machinery.


Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells.

Zolov SN, Lupashin VV - J. Cell Biol. (2005)

The COG complex interacts with retrograde Golgi SNARE GS28 and β-COPI. (A and B) Protein complexes from detergent-solubilized rat liver Golgi were IP using anti-Cog3p (A, lane 1; B, lane 2), anti-GS28 (A, lane 4), preimmune IgGs (A, lane 3), or anti-PDI (B, lane 3). 10% of the Golgi lysates were loaded as a control (A, lane 2; B, lane 1). Note that Cog3p, GS28, and β-COPI were not recovered with control beads or beads loaded with anti-PDI antibodies (A, lane 3; B, lane 3). (C) CCD vesicles are partially COPI coated and do not carry VSVG. COG3 KD cells that express VSVG-GFP were fixed and processed for IF with mouse anti-GS15 antibodies and rabbit anti–ɛ-COPI antibodies as described in Materials and methods. All images were acquired with 63× objective and deconvolved. Note that a number of GS15-labeled CCD vesicles were colabeled with antibodies to ɛ-COPI coat (inset, arrowheads). VSVG was partially colocalized with the GS15 only on a fragmented Golgi membrane (inset, membranes labeled with asterisk), but not on CCD vesicles. Bar, 10 μm. (D) CCD vesicles do not significantly colocalize with the ER. COG3 KD cells were fixed and stained with rabbit anti-Sec61p (red) and mouse anti-GS15 IgGs (green). Bar, 10 μm. (E) CCD vesicles are distinct from early endosomes. COG3 KD cells were fixed and stained with rabbit IgGs to GPP130 (red) and mouse anti-EEA1 IgGs (green). Bar, 10 μm.
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Related In: Results  -  Collection

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fig9: The COG complex interacts with retrograde Golgi SNARE GS28 and β-COPI. (A and B) Protein complexes from detergent-solubilized rat liver Golgi were IP using anti-Cog3p (A, lane 1; B, lane 2), anti-GS28 (A, lane 4), preimmune IgGs (A, lane 3), or anti-PDI (B, lane 3). 10% of the Golgi lysates were loaded as a control (A, lane 2; B, lane 1). Note that Cog3p, GS28, and β-COPI were not recovered with control beads or beads loaded with anti-PDI antibodies (A, lane 3; B, lane 3). (C) CCD vesicles are partially COPI coated and do not carry VSVG. COG3 KD cells that express VSVG-GFP were fixed and processed for IF with mouse anti-GS15 antibodies and rabbit anti–ɛ-COPI antibodies as described in Materials and methods. All images were acquired with 63× objective and deconvolved. Note that a number of GS15-labeled CCD vesicles were colabeled with antibodies to ɛ-COPI coat (inset, arrowheads). VSVG was partially colocalized with the GS15 only on a fragmented Golgi membrane (inset, membranes labeled with asterisk), but not on CCD vesicles. Bar, 10 μm. (D) CCD vesicles do not significantly colocalize with the ER. COG3 KD cells were fixed and stained with rabbit anti-Sec61p (red) and mouse anti-GS15 IgGs (green). Bar, 10 μm. (E) CCD vesicles are distinct from early endosomes. COG3 KD cells were fixed and stained with rabbit IgGs to GPP130 (red) and mouse anti-EEA1 IgGs (green). Bar, 10 μm.
Mentions: We have previously demonstrated that the yeast COG complex regulates intra-Golgi retrograde trafficking and interacts with the essential components of core machinery of vesicular Golgi traffic, i.e., intra-Golgi SNARE proteins and COPI vesicular coat (Suvorova et al., 2002). To test if the mammalian COG complex may also interact with both SNARE and COPI components, we isolated Golgi from the rat liver and performed an IP of the COG complex, v-SNARE GS28, and PDI from detergent-solubilized membranes. WB analysis of coIP proteins revealed that both GS28 (∼5% from total) and small amounts of t-SNARE Syntaxin5 were coprecipitated with the Cog3p (Fig. 9 A, lane 1; unpublished data). It was previously shown that Syntaxin5 and GS28 form a Golgi-localized SNARE complex (Hay et al., 1997) and our findings suggest that the COG complex can interact with either individual SNARE proteins or with the entire SNARE core complex. In the reciprocal precipitation, Cog3p was coIP by anti-GS28 antibodies (Fig. 9 A, lane 4). Neither Cog3p nor the Golgi SNARE molecules were precipitated with control beads (Fig. 9 A, lane 3). We have also determined that the β subunit of the COPI coat was specifically precipitated with anti-Cog3p antibodies (Fig. 9 B, lane 2). These results indicated that the mammalian COG complex like its yeast homologue could physically interact with both vesicular and Golgi-localized components of membrane transport machinery.

Bottom Line: In this work we used short interfering RNA strategy to achieve an efficient knockdown (KD) of Cog3p in HeLa cells.Fragmented Golgi membranes maintained their juxtanuclear localization, cisternal organization and are competent for the anterograde trafficking of vesicular stomatitis virus G protein to the plasma membrane.In a contrast, Cog3p KD resulted in inhibition of retrograde trafficking of the Shiga toxin.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

ABSTRACT
The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved multi-subunit protein complex that regulates membrane trafficking in eukaryotic cells. In this work we used short interfering RNA strategy to achieve an efficient knockdown (KD) of Cog3p in HeLa cells. For the first time, we have demonstrated that Cog3p depletion is accompanied by reduction in Cog1, 2, and 4 protein levels and by accumulation of COG complex-dependent (CCD) vesicles carrying v-SNAREs GS15 and GS28 and cis-Golgi glycoprotein GPP130. Some of these CCD vesicles appeared to be vesicular coat complex I (COPI) coated. A prolonged block in CCD vesicles tethering is accompanied by extensive fragmentation of the Golgi ribbon. Fragmented Golgi membranes maintained their juxtanuclear localization, cisternal organization and are competent for the anterograde trafficking of vesicular stomatitis virus G protein to the plasma membrane. In a contrast, Cog3p KD resulted in inhibition of retrograde trafficking of the Shiga toxin. Furthermore, the mammalian COG complex physically interacts with GS28 and COPI and specifically binds to isolated CCD vesicles.

Show MeSH
Related in: MedlinePlus