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Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block.

Miles S, McManus H, Forsten KE, Storrie B - J. Cell Biol. (2001)

Bottom Line: ER-accumulated Golgi region proteins were functional.Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block.We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.

ABSTRACT
We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1p(dn) concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.

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COPI loses its juxtanuclear Golgi region localization faster than GalNAcT2-GFP in pSARAdn-expressing cells. GalNAcT2-GFP HeLa cells were microinjected with pSARAdn (asterisks) and fixed at 0 t and 1 and 3 h. Cells were stained for COPI using a Cy3 second antibody.
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fig8: COPI loses its juxtanuclear Golgi region localization faster than GalNAcT2-GFP in pSARAdn-expressing cells. GalNAcT2-GFP HeLa cells were microinjected with pSARAdn (asterisks) and fixed at 0 t and 1 and 3 h. Cells were stained for COPI using a Cy3 second antibody.

Mentions: To test if an ER exit block results in the relocation of other cis Golgi matrix proteins with similar kinetics to that of a cisternal Golgi matrix enzyme, HeLa cells were microinjected with pSARAdn and the distribution of endogenous putative matrix or coat proteins observed relative to GalNAcT2-GFP. As shown in Figs. 8–10, the concentrated juxtanuclear distribution of each of the three peripheral proteins decreased decidedly in a time-dependent manner. COPI coat protein relocated rapidly and close to complete loss of juxtanuclear fluorescence was observed after 1h (Fig. 8). Much of this relocation may be to the cytosol. COPI exists as a membrane-associated coat and as soluble coatomer. p115 lost juxtanuclear localization at least as fast as GalNAcT2-GFP (Fig. 9). GM130 relocated with roughly the same kinetics as GalNAcT2-GFP (Fig. 10). To better characterize the site of relocation of these peripheral proteins in HeLa cells, the distribution of GM130 was determined in GalNAcT2-VSV–tagged HeLa cells (Röttger et al., 1998) microinjected with 150 ng/μl stock concentration pSARAdn, incubated for 6 h, and then fixed with methanol. Methanol fixation better preserves microtubules than formaldehyde fixation and as such should give a better preservation of the ER. The distribution of GM130 in the injected cells (Fig. 11 A, asterisks) was predominantly diffuse with some local concentrations. The diffuse labeling may well be cytosolic. GalNAcT2 in the same cells displayed a distinctly ER-like distribution (Fig. 11 B). We conclude that putative Golgi matrix proteins including GM130 relocate, likely in part to the cytosol, with roughly similar kinetics to GalNAcT2 in response to a strong ER exit block.


Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block.

Miles S, McManus H, Forsten KE, Storrie B - J. Cell Biol. (2001)

COPI loses its juxtanuclear Golgi region localization faster than GalNAcT2-GFP in pSARAdn-expressing cells. GalNAcT2-GFP HeLa cells were microinjected with pSARAdn (asterisks) and fixed at 0 t and 1 and 3 h. Cells were stained for COPI using a Cy3 second antibody.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198853&req=5

fig8: COPI loses its juxtanuclear Golgi region localization faster than GalNAcT2-GFP in pSARAdn-expressing cells. GalNAcT2-GFP HeLa cells were microinjected with pSARAdn (asterisks) and fixed at 0 t and 1 and 3 h. Cells were stained for COPI using a Cy3 second antibody.
Mentions: To test if an ER exit block results in the relocation of other cis Golgi matrix proteins with similar kinetics to that of a cisternal Golgi matrix enzyme, HeLa cells were microinjected with pSARAdn and the distribution of endogenous putative matrix or coat proteins observed relative to GalNAcT2-GFP. As shown in Figs. 8–10, the concentrated juxtanuclear distribution of each of the three peripheral proteins decreased decidedly in a time-dependent manner. COPI coat protein relocated rapidly and close to complete loss of juxtanuclear fluorescence was observed after 1h (Fig. 8). Much of this relocation may be to the cytosol. COPI exists as a membrane-associated coat and as soluble coatomer. p115 lost juxtanuclear localization at least as fast as GalNAcT2-GFP (Fig. 9). GM130 relocated with roughly the same kinetics as GalNAcT2-GFP (Fig. 10). To better characterize the site of relocation of these peripheral proteins in HeLa cells, the distribution of GM130 was determined in GalNAcT2-VSV–tagged HeLa cells (Röttger et al., 1998) microinjected with 150 ng/μl stock concentration pSARAdn, incubated for 6 h, and then fixed with methanol. Methanol fixation better preserves microtubules than formaldehyde fixation and as such should give a better preservation of the ER. The distribution of GM130 in the injected cells (Fig. 11 A, asterisks) was predominantly diffuse with some local concentrations. The diffuse labeling may well be cytosolic. GalNAcT2 in the same cells displayed a distinctly ER-like distribution (Fig. 11 B). We conclude that putative Golgi matrix proteins including GM130 relocate, likely in part to the cytosol, with roughly similar kinetics to GalNAcT2 in response to a strong ER exit block.

Bottom Line: ER-accumulated Golgi region proteins were functional.Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block.We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.

ABSTRACT
We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1p(dn) concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.

Show MeSH
Related in: MedlinePlus