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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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Accumulation of CCD vesicles precedes COG3 KD-induced Golgi fragmentation. Control or COG3 KD cells that stably express GalT-GFP were fixed 48 h after siRNA transfection and stained with anti-GS15 and secondary antibodies conjugated with Alexa 594. Images were acquired with 63× objective and deconvolved. Note that the Golgi (arrows) has not yet been fragmented in a subpopulation of the COG3 KD cells, whereas the majority of GS15 was associated with multiple CCD vesicles. Bar, 10 μm.
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fig7: Accumulation of CCD vesicles precedes COG3 KD-induced Golgi fragmentation. Control or COG3 KD cells that stably express GalT-GFP were fixed 48 h after siRNA transfection and stained with anti-GS15 and secondary antibodies conjugated with Alexa 594. Images were acquired with 63× objective and deconvolved. Note that the Golgi (arrows) has not yet been fragmented in a subpopulation of the COG3 KD cells, whereas the majority of GS15 was associated with multiple CCD vesicles. Bar, 10 μm.

Mentions: Does accumulation of CCD vesicles occur before or after COG3 KD-induced Golgi fragmentation? To answer this question we analyzed COG3 KD cells every 12 h after the initial COG3 siRNA transfection. We have found that 48 h after transfection both GS15 (Fig. 7) and GPP130 (unpublished data) were mostly found in CCD vesicles, whereas overall Golgi structure labeled with GalT-GFP has not yet been disturbed in a subpopulation of the COG3 KD cells. These data support the idea that accumulation of CCD vesicles may occur independently and before the Golgi fragmentation.


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

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

Accumulation of CCD vesicles precedes COG3 KD-induced Golgi fragmentation. Control or COG3 KD cells that stably express GalT-GFP were fixed 48 h after siRNA transfection and stained with anti-GS15 and secondary antibodies conjugated with Alexa 594. Images were acquired with 63× objective and deconvolved. Note that the Golgi (arrows) has not yet been fragmented in a subpopulation of the COG3 KD cells, whereas the majority of GS15 was associated with multiple CCD vesicles. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Accumulation of CCD vesicles precedes COG3 KD-induced Golgi fragmentation. Control or COG3 KD cells that stably express GalT-GFP were fixed 48 h after siRNA transfection and stained with anti-GS15 and secondary antibodies conjugated with Alexa 594. Images were acquired with 63× objective and deconvolved. Note that the Golgi (arrows) has not yet been fragmented in a subpopulation of the COG3 KD cells, whereas the majority of GS15 was associated with multiple CCD vesicles. Bar, 10 μm.
Mentions: Does accumulation of CCD vesicles occur before or after COG3 KD-induced Golgi fragmentation? To answer this question we analyzed COG3 KD cells every 12 h after the initial COG3 siRNA transfection. We have found that 48 h after transfection both GS15 (Fig. 7) and GPP130 (unpublished data) were mostly found in CCD vesicles, whereas overall Golgi structure labeled with GalT-GFP has not yet been disturbed in a subpopulation of the COG3 KD cells. These data support the idea that accumulation of CCD vesicles may occur independently and before the 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