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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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COG3 KD results in accumulation of multiple vesicles that carry v-SNAREs GS15, GS28, and cis-Golgi recycling protein GPP130. Control and COG3 KD cells were fixed 72 h after transfection and analyzed by IF using primary antibodies to indicated proteins and appropriate Alexa 488– and Alexa 595–conjugated secondary antibodies. (A) cis-Golgi tether p115, cis/medial Golgi marker giantin, and trans-Golgi tether p230 are present almost exclusively on Golgi ribbon in control cells and on fragmented juxtanuclear Golgi membranes in COG3 KD cells. (B) v-SNAREs GS15, GS28, and cis-Golgi marker GPP130 are Golgi located in control cells and predominantly localized on multiple small structures distributed throughout the COG3 KD cells. Bars, 10 μm.
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fig5: COG3 KD results in accumulation of multiple vesicles that carry v-SNAREs GS15, GS28, and cis-Golgi recycling protein GPP130. Control and COG3 KD cells were fixed 72 h after transfection and analyzed by IF using primary antibodies to indicated proteins and appropriate Alexa 488– and Alexa 595–conjugated secondary antibodies. (A) cis-Golgi tether p115, cis/medial Golgi marker giantin, and trans-Golgi tether p230 are present almost exclusively on Golgi ribbon in control cells and on fragmented juxtanuclear Golgi membranes in COG3 KD cells. (B) v-SNAREs GS15, GS28, and cis-Golgi marker GPP130 are Golgi located in control cells and predominantly localized on multiple small structures distributed throughout the COG3 KD cells. Bars, 10 μm.

Mentions: Detailed IF analysis of different Golgi resident proteins in COG3 KD cells revealed that the majority of integral and peripheral membrane Golgi proteins, including cis-Golgi tethering factors p115 (Nelson et al., 1998) and GM130 (Nakamura et al., 1995), cis-Golgi t-SNARE syntaxin 5 (Hay et al., 1998), cis/medial Golgi tethering protein giantin (Linstedt and Hauri, 1993), and trans-Golgi tether p230 (Brown et al., 2001), were present almost exclusively on relatively large (1–3 μm in size) fragmented Golgi membranes (Fig. 5 A; unpublished data). Surprisingly, we have found that a subset of Golgi proteins, intra-Golgi v-SNAREs GS15 (Xu et al., 2002) and GS28 (Hay et al., 1998), and cis-Golgi phosphoprotein GPP130 (Linstedt et al., 1997) were localized preferentially to multiple small vesicle-like structures distributed throughout the cytoplasm of Cog3p KD cells (Fig. 5 B). In control cells all these three proteins were primarily localized to a juxtanuclear Golgi.


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

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

COG3 KD results in accumulation of multiple vesicles that carry v-SNAREs GS15, GS28, and cis-Golgi recycling protein GPP130. Control and COG3 KD cells were fixed 72 h after transfection and analyzed by IF using primary antibodies to indicated proteins and appropriate Alexa 488– and Alexa 595–conjugated secondary antibodies. (A) cis-Golgi tether p115, cis/medial Golgi marker giantin, and trans-Golgi tether p230 are present almost exclusively on Golgi ribbon in control cells and on fragmented juxtanuclear Golgi membranes in COG3 KD cells. (B) v-SNAREs GS15, GS28, and cis-Golgi marker GPP130 are Golgi located in control cells and predominantly localized on multiple small structures distributed throughout the COG3 KD cells. Bars, 10 μm.
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Related In: Results  -  Collection

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fig5: COG3 KD results in accumulation of multiple vesicles that carry v-SNAREs GS15, GS28, and cis-Golgi recycling protein GPP130. Control and COG3 KD cells were fixed 72 h after transfection and analyzed by IF using primary antibodies to indicated proteins and appropriate Alexa 488– and Alexa 595–conjugated secondary antibodies. (A) cis-Golgi tether p115, cis/medial Golgi marker giantin, and trans-Golgi tether p230 are present almost exclusively on Golgi ribbon in control cells and on fragmented juxtanuclear Golgi membranes in COG3 KD cells. (B) v-SNAREs GS15, GS28, and cis-Golgi marker GPP130 are Golgi located in control cells and predominantly localized on multiple small structures distributed throughout the COG3 KD cells. Bars, 10 μm.
Mentions: Detailed IF analysis of different Golgi resident proteins in COG3 KD cells revealed that the majority of integral and peripheral membrane Golgi proteins, including cis-Golgi tethering factors p115 (Nelson et al., 1998) and GM130 (Nakamura et al., 1995), cis-Golgi t-SNARE syntaxin 5 (Hay et al., 1998), cis/medial Golgi tethering protein giantin (Linstedt and Hauri, 1993), and trans-Golgi tether p230 (Brown et al., 2001), were present almost exclusively on relatively large (1–3 μm in size) fragmented Golgi membranes (Fig. 5 A; unpublished data). Surprisingly, we have found that a subset of Golgi proteins, intra-Golgi v-SNAREs GS15 (Xu et al., 2002) and GS28 (Hay et al., 1998), and cis-Golgi phosphoprotein GPP130 (Linstedt et al., 1997) were localized preferentially to multiple small vesicle-like structures distributed throughout the cytoplasm of Cog3p KD cells (Fig. 5 B). In control cells all these three proteins were primarily localized to a juxtanuclear Golgi.

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