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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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SiRNA-induced COG3 KD is destabilizing Lobe A COG complex subunits. (A) Expression of COG subunits after COG3 KD. WB of cell lysates from control and COG3 KD cells. Average levels of the COG subunits (±SD, n = 4) after 72 h of COG3 KD were determined by quantitative WB, and normalized to mock-transfected cells. (B) Membrane localization of COG complex subunits. WB of membrane (P100) and cytosol (S100) fractions. (C) Cog6p localization. Control and COG3 KD cells that stably express GalT-GFP were fixed and analyzed by three-color IF microscopy after immunostaining with anti-Cog6p. DNA was stained with DAPI. Arrows indicate Golgi or Golgi fragments. Bars, 10 μm.
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fig1: SiRNA-induced COG3 KD is destabilizing Lobe A COG complex subunits. (A) Expression of COG subunits after COG3 KD. WB of cell lysates from control and COG3 KD cells. Average levels of the COG subunits (±SD, n = 4) after 72 h of COG3 KD were determined by quantitative WB, and normalized to mock-transfected cells. (B) Membrane localization of COG complex subunits. WB of membrane (P100) and cytosol (S100) fractions. (C) Cog6p localization. Control and COG3 KD cells that stably express GalT-GFP were fixed and analyzed by three-color IF microscopy after immunostaining with anti-Cog6p. DNA was stained with DAPI. Arrows indicate Golgi or Golgi fragments. Bars, 10 μm.

Mentions: To interfere with the COG complex function in HeLa cells we depleted Cog3p by using RNA interference technique (Elbashir et al., 2001). Three different COG3-specific RNA duplexes were tested initially and one of them (sense, AGACUUGUGCAGUUUAACA) efficiently induced reduction of the Cog3 protein level (Fig. 1 A). The expression of other lobe A COG subunits Cog1p, Cog2p, and Cog4p was also reduced 72 h after COG3 KD, whereas protein level of the lobe B Cog5-8p subunits remained unchanged. Similarly, cellular levels were unchanged for other tested cell proteins: protein disulphide isomerase (PDI), actin, GS28, syntaxin 5, GPP130, and p115 (Fig. 1 and unpublished data). Cell transfection with a nonspecific siRNA did not change the levels of COG subunits expression (unpublished data).


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

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

SiRNA-induced COG3 KD is destabilizing Lobe A COG complex subunits. (A) Expression of COG subunits after COG3 KD. WB of cell lysates from control and COG3 KD cells. Average levels of the COG subunits (±SD, n = 4) after 72 h of COG3 KD were determined by quantitative WB, and normalized to mock-transfected cells. (B) Membrane localization of COG complex subunits. WB of membrane (P100) and cytosol (S100) fractions. (C) Cog6p localization. Control and COG3 KD cells that stably express GalT-GFP were fixed and analyzed by three-color IF microscopy after immunostaining with anti-Cog6p. DNA was stained with DAPI. Arrows indicate Golgi or Golgi fragments. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: SiRNA-induced COG3 KD is destabilizing Lobe A COG complex subunits. (A) Expression of COG subunits after COG3 KD. WB of cell lysates from control and COG3 KD cells. Average levels of the COG subunits (±SD, n = 4) after 72 h of COG3 KD were determined by quantitative WB, and normalized to mock-transfected cells. (B) Membrane localization of COG complex subunits. WB of membrane (P100) and cytosol (S100) fractions. (C) Cog6p localization. Control and COG3 KD cells that stably express GalT-GFP were fixed and analyzed by three-color IF microscopy after immunostaining with anti-Cog6p. DNA was stained with DAPI. Arrows indicate Golgi or Golgi fragments. Bars, 10 μm.
Mentions: To interfere with the COG complex function in HeLa cells we depleted Cog3p by using RNA interference technique (Elbashir et al., 2001). Three different COG3-specific RNA duplexes were tested initially and one of them (sense, AGACUUGUGCAGUUUAACA) efficiently induced reduction of the Cog3 protein level (Fig. 1 A). The expression of other lobe A COG subunits Cog1p, Cog2p, and Cog4p was also reduced 72 h after COG3 KD, whereas protein level of the lobe B Cog5-8p subunits remained unchanged. Similarly, cellular levels were unchanged for other tested cell proteins: protein disulphide isomerase (PDI), actin, GS28, syntaxin 5, GPP130, and p115 (Fig. 1 and unpublished data). Cell transfection with a nonspecific siRNA did not change the levels of COG subunits expression (unpublished data).

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