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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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Characterization of CCD vesicles. (A) Apparent release of GPP130-containing vesicles upon digitonin permeabilization. Control or COG3 KD cells were either treated with buffer (−digitonin) or treated with 0.04 mg/ml digitonin, fixed, and analyzed by IF. Note that disperse vesicle-like staining of GPP130 in COG3 KD cells was eliminated after digitonin treatment. Bars, 10 μl. (B) WB analysis of total cell lysates and subcellular fractions prepared from COG3 KD and mock-treated HeLa cells. Cell lysates were fractionated on heavy membranes (P10) and light vesicle-containing fraction (S10). Equal amount of protein (10 μg) was loaded per lane and analyzed by WB with antibodies to GPP130, GS28, syntaxin 5, and PDI. A portion of marker proteins that was found in a vesicular fraction (n = 2) was determined by semi-quantitative WB. (C) Separation of GPP130-containing membranes by gel filtration. PNS from both control and COG3 KD cells was loaded on a Sephacryl S-1,000 column; 0.5-ml fractions were collected and analyzed by semi-quantitative WB. (D) Distribution of GPP130 and PDI on a velocity gradient. PNS from the COG3 KD cells was loaded on a 10–30% glycerol gradient. GPP130 and PDI were analyzed in fractions by semi-quantitative WB. Fraction 1 corresponds to the top of the gradient. (E) Analysis of CCD vesicle fraction. Fractions 3 and 4 from the glycerol gradient were concentrated by ultracentrifugation. Relative concentrations of Golgi and ER proteins were analyzed by WB as described in Materials and methods. (F) CCD vesicles specifically bind to the COG complex in vitro. PNS from COG3 KD cells was incubated with control beads, with beads loaded with the COG complex (COG), or with anti-GS15 IgGs (α-GS15). Precipitates were analyzed by WB with anti-GPP130 IgGs. (G) Accumulation of GalNAcT2 in CCD vesicles. Control or COG3 KD cells that stably express GalNAcT2-VSV were fixed and stained with mAbs to GPP130 or polyclonal anti–VSV-tag and secondary antibodies conjugated with Alexa 594 (GalNAc-T2-VSV) or Alexa 488 (GPP130) as described in Materials and methods. DNA was stained with DAPI. Images were acquired with 63× objective and deconvolved. Double IF labeling revealed that the vesicular pool of GalNAc-T2-VSV increased significantly in COG3 KD cells. Note that some vesicular profiles were double-labeled with both Golgi markers (insets). Bar, 10 μm.
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fig6: Characterization of CCD vesicles. (A) Apparent release of GPP130-containing vesicles upon digitonin permeabilization. Control or COG3 KD cells were either treated with buffer (−digitonin) or treated with 0.04 mg/ml digitonin, fixed, and analyzed by IF. Note that disperse vesicle-like staining of GPP130 in COG3 KD cells was eliminated after digitonin treatment. Bars, 10 μl. (B) WB analysis of total cell lysates and subcellular fractions prepared from COG3 KD and mock-treated HeLa cells. Cell lysates were fractionated on heavy membranes (P10) and light vesicle-containing fraction (S10). Equal amount of protein (10 μg) was loaded per lane and analyzed by WB with antibodies to GPP130, GS28, syntaxin 5, and PDI. A portion of marker proteins that was found in a vesicular fraction (n = 2) was determined by semi-quantitative WB. (C) Separation of GPP130-containing membranes by gel filtration. PNS from both control and COG3 KD cells was loaded on a Sephacryl S-1,000 column; 0.5-ml fractions were collected and analyzed by semi-quantitative WB. (D) Distribution of GPP130 and PDI on a velocity gradient. PNS from the COG3 KD cells was loaded on a 10–30% glycerol gradient. GPP130 and PDI were analyzed in fractions by semi-quantitative WB. Fraction 1 corresponds to the top of the gradient. (E) Analysis of CCD vesicle fraction. Fractions 3 and 4 from the glycerol gradient were concentrated by ultracentrifugation. Relative concentrations of Golgi and ER proteins were analyzed by WB as described in Materials and methods. (F) CCD vesicles specifically bind to the COG complex in vitro. PNS from COG3 KD cells was incubated with control beads, with beads loaded with the COG complex (COG), or with anti-GS15 IgGs (α-GS15). Precipitates were analyzed by WB with anti-GPP130 IgGs. (G) Accumulation of GalNAcT2 in CCD vesicles. Control or COG3 KD cells that stably express GalNAcT2-VSV were fixed and stained with mAbs to GPP130 or polyclonal anti–VSV-tag and secondary antibodies conjugated with Alexa 594 (GalNAc-T2-VSV) or Alexa 488 (GPP130) as described in Materials and methods. DNA was stained with DAPI. Images were acquired with 63× objective and deconvolved. Double IF labeling revealed that the vesicular pool of GalNAc-T2-VSV increased significantly in COG3 KD cells. Note that some vesicular profiles were double-labeled with both Golgi markers (insets). Bar, 10 μm.

Mentions: Small GPP130-positive structures in COG3 KD cells presumably represent nontethered vesicles, because, in cells permeabilized with the mild detergent digitonin, these structures were efficiently washed away from cells, whereas large Golgi cisternae remained inside the cells (Fig. 6 A). Similar digitonin sensitivity was previously observed for Golgi-derived vesicles that are transiently accumulated during mitosis (Jesch and Linstedt, 1998).


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

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

Characterization of CCD vesicles. (A) Apparent release of GPP130-containing vesicles upon digitonin permeabilization. Control or COG3 KD cells were either treated with buffer (−digitonin) or treated with 0.04 mg/ml digitonin, fixed, and analyzed by IF. Note that disperse vesicle-like staining of GPP130 in COG3 KD cells was eliminated after digitonin treatment. Bars, 10 μl. (B) WB analysis of total cell lysates and subcellular fractions prepared from COG3 KD and mock-treated HeLa cells. Cell lysates were fractionated on heavy membranes (P10) and light vesicle-containing fraction (S10). Equal amount of protein (10 μg) was loaded per lane and analyzed by WB with antibodies to GPP130, GS28, syntaxin 5, and PDI. A portion of marker proteins that was found in a vesicular fraction (n = 2) was determined by semi-quantitative WB. (C) Separation of GPP130-containing membranes by gel filtration. PNS from both control and COG3 KD cells was loaded on a Sephacryl S-1,000 column; 0.5-ml fractions were collected and analyzed by semi-quantitative WB. (D) Distribution of GPP130 and PDI on a velocity gradient. PNS from the COG3 KD cells was loaded on a 10–30% glycerol gradient. GPP130 and PDI were analyzed in fractions by semi-quantitative WB. Fraction 1 corresponds to the top of the gradient. (E) Analysis of CCD vesicle fraction. Fractions 3 and 4 from the glycerol gradient were concentrated by ultracentrifugation. Relative concentrations of Golgi and ER proteins were analyzed by WB as described in Materials and methods. (F) CCD vesicles specifically bind to the COG complex in vitro. PNS from COG3 KD cells was incubated with control beads, with beads loaded with the COG complex (COG), or with anti-GS15 IgGs (α-GS15). Precipitates were analyzed by WB with anti-GPP130 IgGs. (G) Accumulation of GalNAcT2 in CCD vesicles. Control or COG3 KD cells that stably express GalNAcT2-VSV were fixed and stained with mAbs to GPP130 or polyclonal anti–VSV-tag and secondary antibodies conjugated with Alexa 594 (GalNAc-T2-VSV) or Alexa 488 (GPP130) as described in Materials and methods. DNA was stained with DAPI. Images were acquired with 63× objective and deconvolved. Double IF labeling revealed that the vesicular pool of GalNAc-T2-VSV increased significantly in COG3 KD cells. Note that some vesicular profiles were double-labeled with both Golgi markers (insets). Bar, 10 μm.
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Related In: Results  -  Collection

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fig6: Characterization of CCD vesicles. (A) Apparent release of GPP130-containing vesicles upon digitonin permeabilization. Control or COG3 KD cells were either treated with buffer (−digitonin) or treated with 0.04 mg/ml digitonin, fixed, and analyzed by IF. Note that disperse vesicle-like staining of GPP130 in COG3 KD cells was eliminated after digitonin treatment. Bars, 10 μl. (B) WB analysis of total cell lysates and subcellular fractions prepared from COG3 KD and mock-treated HeLa cells. Cell lysates were fractionated on heavy membranes (P10) and light vesicle-containing fraction (S10). Equal amount of protein (10 μg) was loaded per lane and analyzed by WB with antibodies to GPP130, GS28, syntaxin 5, and PDI. A portion of marker proteins that was found in a vesicular fraction (n = 2) was determined by semi-quantitative WB. (C) Separation of GPP130-containing membranes by gel filtration. PNS from both control and COG3 KD cells was loaded on a Sephacryl S-1,000 column; 0.5-ml fractions were collected and analyzed by semi-quantitative WB. (D) Distribution of GPP130 and PDI on a velocity gradient. PNS from the COG3 KD cells was loaded on a 10–30% glycerol gradient. GPP130 and PDI were analyzed in fractions by semi-quantitative WB. Fraction 1 corresponds to the top of the gradient. (E) Analysis of CCD vesicle fraction. Fractions 3 and 4 from the glycerol gradient were concentrated by ultracentrifugation. Relative concentrations of Golgi and ER proteins were analyzed by WB as described in Materials and methods. (F) CCD vesicles specifically bind to the COG complex in vitro. PNS from COG3 KD cells was incubated with control beads, with beads loaded with the COG complex (COG), or with anti-GS15 IgGs (α-GS15). Precipitates were analyzed by WB with anti-GPP130 IgGs. (G) Accumulation of GalNAcT2 in CCD vesicles. Control or COG3 KD cells that stably express GalNAcT2-VSV were fixed and stained with mAbs to GPP130 or polyclonal anti–VSV-tag and secondary antibodies conjugated with Alexa 594 (GalNAc-T2-VSV) or Alexa 488 (GPP130) as described in Materials and methods. DNA was stained with DAPI. Images were acquired with 63× objective and deconvolved. Double IF labeling revealed that the vesicular pool of GalNAc-T2-VSV increased significantly in COG3 KD cells. Note that some vesicular profiles were double-labeled with both Golgi markers (insets). Bar, 10 μm.
Mentions: Small GPP130-positive structures in COG3 KD cells presumably represent nontethered vesicles, because, in cells permeabilized with the mild detergent digitonin, these structures were efficiently washed away from cells, whereas large Golgi cisternae remained inside the cells (Fig. 6 A). Similar digitonin sensitivity was previously observed for Golgi-derived vesicles that are transiently accumulated during mitosis (Jesch and Linstedt, 1998).

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