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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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Golgi in COG3 KD cells is disrupted to mini-stacks, which are proficient in anterograde VSVG delivery to plasma membrane and defective in retrograde trafficking of Shiga toxin B subunit. (A) Control and COG3 KD cells that stably express GalNAcT2-GFP were fixed and stained with anti-p115, anti-giantin, or anti-p230 antibodies and secondary antibodies conjugated with Alexa 594. DNA was stained with DAPI. Images were acquired with 100× objective and deconvolved. Bar, 10 μm. (B) Ultrastructural analysis of Golgi in COG3 KD cells. Electron micrographs of the juxtanuclear region in control (i) and COG3 KD (ii and iii) cells. Note the Golgi mini-stack in ii and multiple 60-nm vesicles in COG3 KD cells (ii and iii, arrows). G, Golgi; M, mitochondria; N, nucleus; ER, endoplasmic reticulum. Bars, 1 μm. (C) Control and COG3 KD cells were transfected with the VSVG-GFP-ts045 vector. VSVG was accumulated in the ER for 16 h at 39.5°C. After that cells were transferred to 32°C, incubated for 2 h, fixed, and processed for IF with anti-GS28 antibodies. Both control and COG3 KD cells accumulated VSVG-GFP on the cell surface (merged images, arrows). Some VSVG was also found on GS28-positive Golgi membranes in control cells and on juxtanuclear Golgi fragments in COG3 KD cells. The major pool of GS28 was localized on a VSVG-GFP-negative CCD vesicles in COG3 KD cells (inset). Bar, 10 μm. (D) ∼100 cells in both control and COG3 KD samples were analyzed and each cell was assigned in specific group bases on VSVG localization profile. PM, VSVG localized only on the plasma membrane; PM+Golgi, VSVG localized mostly on the plasma membrane, but partially (<30%) on the Golgi; Golgi+PM, VSVG localized on the plasma membrane, but mostly on the Golgi; and ER+Golgi, accumulation of the VSVG in the ER. All images were acquired with 63× objective and deconvolved. (E) Retrograde trafficking of STB-Cy3. Control and COG3 KD cells that stably express GalT-GFP were pulse incubated with the STB-Cy3 as described in Materials and methods and STB was allowed to internalize for 2 h. Cells were fixed and ER was visualized with ER-Tracker. Note that majority of STB in control cells reached the Golgi (arrows), whereas in COG3 KD cells the STB-Cy3 signal was detected only on cell periphery. Bars, 10 μm. (F) Same as in E, except GalNAc-T2-GFP HeLa cells were used and STB-Cy3 was internalized for 12 h. Bars, 10 μm.
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fig8: Golgi in COG3 KD cells is disrupted to mini-stacks, which are proficient in anterograde VSVG delivery to plasma membrane and defective in retrograde trafficking of Shiga toxin B subunit. (A) Control and COG3 KD cells that stably express GalNAcT2-GFP were fixed and stained with anti-p115, anti-giantin, or anti-p230 antibodies and secondary antibodies conjugated with Alexa 594. DNA was stained with DAPI. Images were acquired with 100× objective and deconvolved. Bar, 10 μm. (B) Ultrastructural analysis of Golgi in COG3 KD cells. Electron micrographs of the juxtanuclear region in control (i) and COG3 KD (ii and iii) cells. Note the Golgi mini-stack in ii and multiple 60-nm vesicles in COG3 KD cells (ii and iii, arrows). G, Golgi; M, mitochondria; N, nucleus; ER, endoplasmic reticulum. Bars, 1 μm. (C) Control and COG3 KD cells were transfected with the VSVG-GFP-ts045 vector. VSVG was accumulated in the ER for 16 h at 39.5°C. After that cells were transferred to 32°C, incubated for 2 h, fixed, and processed for IF with anti-GS28 antibodies. Both control and COG3 KD cells accumulated VSVG-GFP on the cell surface (merged images, arrows). Some VSVG was also found on GS28-positive Golgi membranes in control cells and on juxtanuclear Golgi fragments in COG3 KD cells. The major pool of GS28 was localized on a VSVG-GFP-negative CCD vesicles in COG3 KD cells (inset). Bar, 10 μm. (D) ∼100 cells in both control and COG3 KD samples were analyzed and each cell was assigned in specific group bases on VSVG localization profile. PM, VSVG localized only on the plasma membrane; PM+Golgi, VSVG localized mostly on the plasma membrane, but partially (<30%) on the Golgi; Golgi+PM, VSVG localized on the plasma membrane, but mostly on the Golgi; and ER+Golgi, accumulation of the VSVG in the ER. All images were acquired with 63× objective and deconvolved. (E) Retrograde trafficking of STB-Cy3. Control and COG3 KD cells that stably express GalT-GFP were pulse incubated with the STB-Cy3 as described in Materials and methods and STB was allowed to internalize for 2 h. Cells were fixed and ER was visualized with ER-Tracker. Note that majority of STB in control cells reached the Golgi (arrows), whereas in COG3 KD cells the STB-Cy3 signal was detected only on cell periphery. Bars, 10 μm. (F) Same as in E, except GalNAc-T2-GFP HeLa cells were used and STB-Cy3 was internalized for 12 h. Bars, 10 μm.

Mentions: Although siRNA-induced COG3 KD induced both accumulation of CCD vesicles and Golgi fragmentation, cells were able to multiply and their growth rate was not severely affected (unpublished data). Moreover, fragmented Golgi membranes maintained their juxtanuclear localization (Fig. 5 A). Detailed IF analysis revealed that cis-Golgi p115 (Nelson et al., 1998), and the medial Golgi GalNAc-T2 (Storrie et al., 1998), maintained their overlapping but distinct distribution on both control Golgi ribbon-like structure and Golgi fragments in COG3 KD cells (Fig. 8 A, GalNacT2/p115 frames). Similarly, localization of two medial Golgi proteins GalNAcT2 and giantin (Linstedt and Hauri, 1993) almost completely overlapped in both control and COG3 KD-treated cells (Fig. 8 A, GalNacT2/Giantin frames). And finally trans-Golgi localized p230 (Brown et al., 2001) maintained its relative localization in COG3 KD cells (Fig. 8 A, GalNacT2/p230 frames). We concluded that Cog3p KD resulted in Golgi fragmentation into multiple mini-Golgi stacks.


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

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

Golgi in COG3 KD cells is disrupted to mini-stacks, which are proficient in anterograde VSVG delivery to plasma membrane and defective in retrograde trafficking of Shiga toxin B subunit. (A) Control and COG3 KD cells that stably express GalNAcT2-GFP were fixed and stained with anti-p115, anti-giantin, or anti-p230 antibodies and secondary antibodies conjugated with Alexa 594. DNA was stained with DAPI. Images were acquired with 100× objective and deconvolved. Bar, 10 μm. (B) Ultrastructural analysis of Golgi in COG3 KD cells. Electron micrographs of the juxtanuclear region in control (i) and COG3 KD (ii and iii) cells. Note the Golgi mini-stack in ii and multiple 60-nm vesicles in COG3 KD cells (ii and iii, arrows). G, Golgi; M, mitochondria; N, nucleus; ER, endoplasmic reticulum. Bars, 1 μm. (C) Control and COG3 KD cells were transfected with the VSVG-GFP-ts045 vector. VSVG was accumulated in the ER for 16 h at 39.5°C. After that cells were transferred to 32°C, incubated for 2 h, fixed, and processed for IF with anti-GS28 antibodies. Both control and COG3 KD cells accumulated VSVG-GFP on the cell surface (merged images, arrows). Some VSVG was also found on GS28-positive Golgi membranes in control cells and on juxtanuclear Golgi fragments in COG3 KD cells. The major pool of GS28 was localized on a VSVG-GFP-negative CCD vesicles in COG3 KD cells (inset). Bar, 10 μm. (D) ∼100 cells in both control and COG3 KD samples were analyzed and each cell was assigned in specific group bases on VSVG localization profile. PM, VSVG localized only on the plasma membrane; PM+Golgi, VSVG localized mostly on the plasma membrane, but partially (<30%) on the Golgi; Golgi+PM, VSVG localized on the plasma membrane, but mostly on the Golgi; and ER+Golgi, accumulation of the VSVG in the ER. All images were acquired with 63× objective and deconvolved. (E) Retrograde trafficking of STB-Cy3. Control and COG3 KD cells that stably express GalT-GFP were pulse incubated with the STB-Cy3 as described in Materials and methods and STB was allowed to internalize for 2 h. Cells were fixed and ER was visualized with ER-Tracker. Note that majority of STB in control cells reached the Golgi (arrows), whereas in COG3 KD cells the STB-Cy3 signal was detected only on cell periphery. Bars, 10 μm. (F) Same as in E, except GalNAc-T2-GFP HeLa cells were used and STB-Cy3 was internalized for 12 h. Bars, 10 μm.
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fig8: Golgi in COG3 KD cells is disrupted to mini-stacks, which are proficient in anterograde VSVG delivery to plasma membrane and defective in retrograde trafficking of Shiga toxin B subunit. (A) Control and COG3 KD cells that stably express GalNAcT2-GFP were fixed and stained with anti-p115, anti-giantin, or anti-p230 antibodies and secondary antibodies conjugated with Alexa 594. DNA was stained with DAPI. Images were acquired with 100× objective and deconvolved. Bar, 10 μm. (B) Ultrastructural analysis of Golgi in COG3 KD cells. Electron micrographs of the juxtanuclear region in control (i) and COG3 KD (ii and iii) cells. Note the Golgi mini-stack in ii and multiple 60-nm vesicles in COG3 KD cells (ii and iii, arrows). G, Golgi; M, mitochondria; N, nucleus; ER, endoplasmic reticulum. Bars, 1 μm. (C) Control and COG3 KD cells were transfected with the VSVG-GFP-ts045 vector. VSVG was accumulated in the ER for 16 h at 39.5°C. After that cells were transferred to 32°C, incubated for 2 h, fixed, and processed for IF with anti-GS28 antibodies. Both control and COG3 KD cells accumulated VSVG-GFP on the cell surface (merged images, arrows). Some VSVG was also found on GS28-positive Golgi membranes in control cells and on juxtanuclear Golgi fragments in COG3 KD cells. The major pool of GS28 was localized on a VSVG-GFP-negative CCD vesicles in COG3 KD cells (inset). Bar, 10 μm. (D) ∼100 cells in both control and COG3 KD samples were analyzed and each cell was assigned in specific group bases on VSVG localization profile. PM, VSVG localized only on the plasma membrane; PM+Golgi, VSVG localized mostly on the plasma membrane, but partially (<30%) on the Golgi; Golgi+PM, VSVG localized on the plasma membrane, but mostly on the Golgi; and ER+Golgi, accumulation of the VSVG in the ER. All images were acquired with 63× objective and deconvolved. (E) Retrograde trafficking of STB-Cy3. Control and COG3 KD cells that stably express GalT-GFP were pulse incubated with the STB-Cy3 as described in Materials and methods and STB was allowed to internalize for 2 h. Cells were fixed and ER was visualized with ER-Tracker. Note that majority of STB in control cells reached the Golgi (arrows), whereas in COG3 KD cells the STB-Cy3 signal was detected only on cell periphery. Bars, 10 μm. (F) Same as in E, except GalNAc-T2-GFP HeLa cells were used and STB-Cy3 was internalized for 12 h. Bars, 10 μm.
Mentions: Although siRNA-induced COG3 KD induced both accumulation of CCD vesicles and Golgi fragmentation, cells were able to multiply and their growth rate was not severely affected (unpublished data). Moreover, fragmented Golgi membranes maintained their juxtanuclear localization (Fig. 5 A). Detailed IF analysis revealed that cis-Golgi p115 (Nelson et al., 1998), and the medial Golgi GalNAc-T2 (Storrie et al., 1998), maintained their overlapping but distinct distribution on both control Golgi ribbon-like structure and Golgi fragments in COG3 KD cells (Fig. 8 A, GalNacT2/p115 frames). Similarly, localization of two medial Golgi proteins GalNAcT2 and giantin (Linstedt and Hauri, 1993) almost completely overlapped in both control and COG3 KD-treated cells (Fig. 8 A, GalNacT2/Giantin frames). And finally trans-Golgi localized p230 (Brown et al., 2001) maintained its relative localization in COG3 KD cells (Fig. 8 A, GalNacT2/p230 frames). We concluded that Cog3p KD resulted in Golgi fragmentation into multiple mini-Golgi stacks.

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