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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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Microinjection of anti-Cog3 antibodies disrupts Golgi structure. GalT-GFP HeLa cells were microinjected with anti-Cog3p IgGs and imaged immediately (0 min) or 4 h (240 min) after the injection. Texas red was used as an injection marker. Note that in cells injected with anti-Cog3 IgGs Golgi (labeled with asterisks) become fragmented. Microinjections with the preimmune IgGs did not result in Golgi fragmentation (date not depicted).
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fig4: Microinjection of anti-Cog3 antibodies disrupts Golgi structure. GalT-GFP HeLa cells were microinjected with anti-Cog3p IgGs and imaged immediately (0 min) or 4 h (240 min) after the injection. Texas red was used as an injection marker. Note that in cells injected with anti-Cog3 IgGs Golgi (labeled with asterisks) become fragmented. Microinjections with the preimmune IgGs did not result in Golgi fragmentation (date not depicted).

Mentions: To independently confirm an essential role for the COG complex in Golgi structure maintenance, we used affinity-purified antibodies against Cog3p (Suvorova et al., 2001). These antibodies were previously used for both IF and IP of the endogenous COG complex in mammalian cells (Suvorova et al., 2001; Ungar et al., 2002). We rationalized that upon microinjection into cells anti-Cog3p IgGs would specifically bind to the Cog3p and interfere with the COG complex function. Indeed, we have observed that 4 h after the antibodies microinjection the Golgi ribbon structure was converted into multiple mostly juxtanuclear localized small fragments (Fig. 4). This Golgi phenotype was persistent for up to 20 h after antibody microinjection without any visible signs of cell death. Microinjection with control antibodies did not change the morphology of the Golgi (unpublished data). We have concluded that anti-Cog3p antibodies like the COG3 siRNA act by blocking Cog3p function, which is necessary for the Golgi structure maintenance.


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

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

Microinjection of anti-Cog3 antibodies disrupts Golgi structure. GalT-GFP HeLa cells were microinjected with anti-Cog3p IgGs and imaged immediately (0 min) or 4 h (240 min) after the injection. Texas red was used as an injection marker. Note that in cells injected with anti-Cog3 IgGs Golgi (labeled with asterisks) become fragmented. Microinjections with the preimmune IgGs did not result in Golgi fragmentation (date not depicted).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Microinjection of anti-Cog3 antibodies disrupts Golgi structure. GalT-GFP HeLa cells were microinjected with anti-Cog3p IgGs and imaged immediately (0 min) or 4 h (240 min) after the injection. Texas red was used as an injection marker. Note that in cells injected with anti-Cog3 IgGs Golgi (labeled with asterisks) become fragmented. Microinjections with the preimmune IgGs did not result in Golgi fragmentation (date not depicted).
Mentions: To independently confirm an essential role for the COG complex in Golgi structure maintenance, we used affinity-purified antibodies against Cog3p (Suvorova et al., 2001). These antibodies were previously used for both IF and IP of the endogenous COG complex in mammalian cells (Suvorova et al., 2001; Ungar et al., 2002). We rationalized that upon microinjection into cells anti-Cog3p IgGs would specifically bind to the Cog3p and interfere with the COG complex function. Indeed, we have observed that 4 h after the antibodies microinjection the Golgi ribbon structure was converted into multiple mostly juxtanuclear localized small fragments (Fig. 4). This Golgi phenotype was persistent for up to 20 h after antibody microinjection without any visible signs of cell death. Microinjection with control antibodies did not change the morphology of the Golgi (unpublished data). We have concluded that anti-Cog3p antibodies like the COG3 siRNA act by blocking Cog3p function, which is necessary for the Golgi structure maintenance.

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