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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: Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics.ER-accumulated Golgi region proteins were functional.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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Confocal fluorescence microscopy colocalizes giantin with GalNAcT2 in the ER of cells expressing pSARAdn. HeLa cells stably transfected with GalNAcT2-GFP were microinjected with pSARAdn. After a 6 h expression period, cells were fixed and stained for giantin using a Cy3-conjugated second antibody. A, giantin; B, GalNAcT2-GFP; C, overlay. As shown by the overlay, giantin and GalNAcT2 appeared to extensively colocalize to the nuclear envelope and elsewhere within the cytoplasm. Such localizations are characteristic of the ER. Asterisk (A) indicates microinjected cell.
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fig2: Confocal fluorescence microscopy colocalizes giantin with GalNAcT2 in the ER of cells expressing pSARAdn. HeLa cells stably transfected with GalNAcT2-GFP were microinjected with pSARAdn. After a 6 h expression period, cells were fixed and stained for giantin using a Cy3-conjugated second antibody. A, giantin; B, GalNAcT2-GFP; C, overlay. As shown by the overlay, giantin and GalNAcT2 appeared to extensively colocalize to the nuclear envelope and elsewhere within the cytoplasm. Such localizations are characteristic of the ER. Asterisk (A) indicates microinjected cell.

Mentions: We next determined the dependence of giantin and GalNAcT2 redistribution on the concentration of microinjected pSARAdn plasmid. We had postulated that the plasmid-driven expression of Sar1pdn should be a more effective tool in producing relocation of Golgi region proteins. As shown in Fig. 1, D–F, this proved to be the case, and at high pSARAdn concentrations both GalNAcT2 and giantin redistributed to a similar extent. By fluorescence, the distribution of GalNAcT2 and giantin were similar following a 6-h incubation of cells microinjected with a high concentration of pSARAdn plasmid. Both appeared to have a distribution suggestive of accumulation in the ER. To provide further evidence of ER accumulation, giantin was colocalized relative to GalNAcT2-GFP by confocal fluorescence microscopy. GalNAcT2-GFP accumulates in the ER in correspondence with protein disulfide isomerase in response to an ER exit block (Storrie et al., 2000). As shown by confocal fluorescence microscopy in Fig. 2, giantin in pSARAdn-microinjected cells appeared to rim the cell nucleus in a somewhat discontinuous pattern and to be present in general correspondence to GalNAcT2-GFP at both the nuclear rim and in the cytoplasm. There was extensive overlap between the two in computer overlays. As further evidence and as a biochemical assay of ER accumulation of the reference protein, GalNAcT2, a Golgi apparatus–specific O-glycosylation enzyme, the in situ glycosylation state of an artificial substrate, CD8, fused with the ER retention domain of glucuronyltransferase was characterized. This chimeric protein localizes to the ER and is a potential substrate for in situ O-glycosylation by redistributed Golgi region enzymes. When Sar1pdn-microinjected CD8 glucuronyltransferase cells were reacted with rhodamine-conjugated Helix pomatia lectin, a lectin specific for O-linked terminal α-GalNAc, considerable ER-specific staining was observed 6 h after pSARAdn microinjection (Fig. 3, compare microinjected cells [asterisks] with noninjected cells [arrowheads]), a result indicative of in situ ER activity of Golgi apparatus–specific O-glycosylation enzymes. Note that Helix pomatia–specific staining was restricted to a juxtanuclear, Golgi apparatus–like distribution in the control, non-pSARAdn–injected cells.


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)

Confocal fluorescence microscopy colocalizes giantin with GalNAcT2 in the ER of cells expressing pSARAdn. HeLa cells stably transfected with GalNAcT2-GFP were microinjected with pSARAdn. After a 6 h expression period, cells were fixed and stained for giantin using a Cy3-conjugated second antibody. A, giantin; B, GalNAcT2-GFP; C, overlay. As shown by the overlay, giantin and GalNAcT2 appeared to extensively colocalize to the nuclear envelope and elsewhere within the cytoplasm. Such localizations are characteristic of the ER. Asterisk (A) indicates microinjected cell.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198853&req=5

fig2: Confocal fluorescence microscopy colocalizes giantin with GalNAcT2 in the ER of cells expressing pSARAdn. HeLa cells stably transfected with GalNAcT2-GFP were microinjected with pSARAdn. After a 6 h expression period, cells were fixed and stained for giantin using a Cy3-conjugated second antibody. A, giantin; B, GalNAcT2-GFP; C, overlay. As shown by the overlay, giantin and GalNAcT2 appeared to extensively colocalize to the nuclear envelope and elsewhere within the cytoplasm. Such localizations are characteristic of the ER. Asterisk (A) indicates microinjected cell.
Mentions: We next determined the dependence of giantin and GalNAcT2 redistribution on the concentration of microinjected pSARAdn plasmid. We had postulated that the plasmid-driven expression of Sar1pdn should be a more effective tool in producing relocation of Golgi region proteins. As shown in Fig. 1, D–F, this proved to be the case, and at high pSARAdn concentrations both GalNAcT2 and giantin redistributed to a similar extent. By fluorescence, the distribution of GalNAcT2 and giantin were similar following a 6-h incubation of cells microinjected with a high concentration of pSARAdn plasmid. Both appeared to have a distribution suggestive of accumulation in the ER. To provide further evidence of ER accumulation, giantin was colocalized relative to GalNAcT2-GFP by confocal fluorescence microscopy. GalNAcT2-GFP accumulates in the ER in correspondence with protein disulfide isomerase in response to an ER exit block (Storrie et al., 2000). As shown by confocal fluorescence microscopy in Fig. 2, giantin in pSARAdn-microinjected cells appeared to rim the cell nucleus in a somewhat discontinuous pattern and to be present in general correspondence to GalNAcT2-GFP at both the nuclear rim and in the cytoplasm. There was extensive overlap between the two in computer overlays. As further evidence and as a biochemical assay of ER accumulation of the reference protein, GalNAcT2, a Golgi apparatus–specific O-glycosylation enzyme, the in situ glycosylation state of an artificial substrate, CD8, fused with the ER retention domain of glucuronyltransferase was characterized. This chimeric protein localizes to the ER and is a potential substrate for in situ O-glycosylation by redistributed Golgi region enzymes. When Sar1pdn-microinjected CD8 glucuronyltransferase cells were reacted with rhodamine-conjugated Helix pomatia lectin, a lectin specific for O-linked terminal α-GalNAc, considerable ER-specific staining was observed 6 h after pSARAdn microinjection (Fig. 3, compare microinjected cells [asterisks] with noninjected cells [arrowheads]), a result indicative of in situ ER activity of Golgi apparatus–specific O-glycosylation enzymes. Note that Helix pomatia–specific staining was restricted to a juxtanuclear, Golgi apparatus–like distribution in the control, non-pSARAdn–injected cells.

Bottom Line: Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics.ER-accumulated Golgi region proteins were functional.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