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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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Mouse COG3p expression partially suppresses hCOG3 KD-induced Golgi fragmentation. (A) DNA alignment of a portion of the hCOG3 and mCOG3 sequences. siRNA target region is highlighted in the box. (B) GalT-GFP HeLa cells were either mock transfected (control), transfected with COG3 siRNA (COG3 KD), or simultaneously transfected with COG3p siRNA and mCOG3-encoding plasmid (COG3 KD +mCOG3) and imaged 72 h after transfection. Bar, 10 μm.
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fig3: Mouse COG3p expression partially suppresses hCOG3 KD-induced Golgi fragmentation. (A) DNA alignment of a portion of the hCOG3 and mCOG3 sequences. siRNA target region is highlighted in the box. (B) GalT-GFP HeLa cells were either mock transfected (control), transfected with COG3 siRNA (COG3 KD), or simultaneously transfected with COG3p siRNA and mCOG3-encoding plasmid (COG3 KD +mCOG3) and imaged 72 h after transfection. Bar, 10 μm.

Mentions: To test the specificity of COG3 siRNA KD, we took advantage of the fact that human and mouse Cog3 proteins show 95% identity and, therefore, most likely would functionally substitute each other. In the same time, human and mouse COG3 siRNA target region share only 74% of homology with five miss-matched nucleotides (Fig. 3 A) and this difference can be used in gene-replacement siRNA experiments (Puthenveedu and Linstedt, 2004). In good agreement with our prediction, in HeLa cells, that were cotransfected with both hCOG3 siRNA and the plasmid that expressed mouse Cog3p, the Golgi fragmentation was completely or partially prevented (Fig. 3 B). Similar results were obtained when cells were transfected with mCOG3-containing vector 24 h after hCOG3 siRNA transfection (unpublished data). We concluded that the COG3 KD is specific and that the Cog3p depletion directly induces a reversible Golgi fragmentation.


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

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

Mouse COG3p expression partially suppresses hCOG3 KD-induced Golgi fragmentation. (A) DNA alignment of a portion of the hCOG3 and mCOG3 sequences. siRNA target region is highlighted in the box. (B) GalT-GFP HeLa cells were either mock transfected (control), transfected with COG3 siRNA (COG3 KD), or simultaneously transfected with COG3p siRNA and mCOG3-encoding plasmid (COG3 KD +mCOG3) and imaged 72 h after transfection. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Mouse COG3p expression partially suppresses hCOG3 KD-induced Golgi fragmentation. (A) DNA alignment of a portion of the hCOG3 and mCOG3 sequences. siRNA target region is highlighted in the box. (B) GalT-GFP HeLa cells were either mock transfected (control), transfected with COG3 siRNA (COG3 KD), or simultaneously transfected with COG3p siRNA and mCOG3-encoding plasmid (COG3 KD +mCOG3) and imaged 72 h after transfection. Bar, 10 μm.
Mentions: To test the specificity of COG3 siRNA KD, we took advantage of the fact that human and mouse Cog3 proteins show 95% identity and, therefore, most likely would functionally substitute each other. In the same time, human and mouse COG3 siRNA target region share only 74% of homology with five miss-matched nucleotides (Fig. 3 A) and this difference can be used in gene-replacement siRNA experiments (Puthenveedu and Linstedt, 2004). In good agreement with our prediction, in HeLa cells, that were cotransfected with both hCOG3 siRNA and the plasmid that expressed mouse Cog3p, the Golgi fragmentation was completely or partially prevented (Fig. 3 B). Similar results were obtained when cells were transfected with mCOG3-containing vector 24 h after hCOG3 siRNA transfection (unpublished data). We concluded that the COG3 KD is specific and that the Cog3p depletion directly induces a reversible Golgi fragmentation.

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