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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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Cog3p depletion induces Golgi fragmentation. GalT-GFP HeLa cells were transfected with COG3 siRNA (right column) or mock transfected (left column). 72 h after transfection, cells were fixed and processed for IF with anti-Cog3p (COG3p row), and anti-GM130 (GM130 row) antibodies. The bottom row represent merged three-color images. Bars, 10 μm.
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fig2: Cog3p depletion induces Golgi fragmentation. GalT-GFP HeLa cells were transfected with COG3 siRNA (right column) or mock transfected (left column). 72 h after transfection, cells were fixed and processed for IF with anti-Cog3p (COG3p row), and anti-GM130 (GM130 row) antibodies. The bottom row represent merged three-color images. Bars, 10 μm.

Mentions: Because the level of the lobe B COG subunits was not affected by the Cog3p depletion we have determined their intracellular localization by both immunofluorescence (IF) and Western blot (WB) assays. We have found that significant amounts of both Cog5p and Cog8p were still associated with the membrane fraction (Fig. 1 B). IF analysis of COG3 KD cells revealed that Cog6p (Fig. 1 C) and other lobe B subunits (unpublished data) were localized on large structures in juxtanuclear region that were colocalized with resident Golgi enzyme GFP-tagged β 1,4-galactosyltransferase (GalT-GFP; Fig. 1 C). Detailed analysis of COG3 KD cells revealed that both GalT-GFP and a Golgi tethering factor GM130 were found on fragmented Golgi membranes (Fig. 2).


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

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

Cog3p depletion induces Golgi fragmentation. GalT-GFP HeLa cells were transfected with COG3 siRNA (right column) or mock transfected (left column). 72 h after transfection, cells were fixed and processed for IF with anti-Cog3p (COG3p row), and anti-GM130 (GM130 row) antibodies. The bottom row represent merged three-color images. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171815&req=5

fig2: Cog3p depletion induces Golgi fragmentation. GalT-GFP HeLa cells were transfected with COG3 siRNA (right column) or mock transfected (left column). 72 h after transfection, cells were fixed and processed for IF with anti-Cog3p (COG3p row), and anti-GM130 (GM130 row) antibodies. The bottom row represent merged three-color images. Bars, 10 μm.
Mentions: Because the level of the lobe B COG subunits was not affected by the Cog3p depletion we have determined their intracellular localization by both immunofluorescence (IF) and Western blot (WB) assays. We have found that significant amounts of both Cog5p and Cog8p were still associated with the membrane fraction (Fig. 1 B). IF analysis of COG3 KD cells revealed that Cog6p (Fig. 1 C) and other lobe B subunits (unpublished data) were localized on large structures in juxtanuclear region that were colocalized with resident Golgi enzyme GFP-tagged β 1,4-galactosyltransferase (GalT-GFP; Fig. 1 C). Detailed analysis of COG3 KD cells revealed that both GalT-GFP and a Golgi tethering factor GM130 were found on fragmented Golgi membranes (Fig. 2).

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