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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 - PubMed

  • ️Mon Jan 01 2001

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

S Miles et al. J Cell Biol. 2001.

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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Figures

Figure 1.
Figure 1.

Concentration dependence of the Sar1pdn induced redistribution of GalNAcT2, giantin, and GM130. HeLa cells stably expressing GalNAcT2-GFP were microinjected with either various stock concentrations of Sar1pdn protein (A–C) or pSARAdn plasmid (D–F), incubated for 6 h at 37°C, and then fixed. In the case of cells microinjected with Sar1pdn, the postinjection incubation was in the presence of CHX. Following fixation, cells were stained for either GM130 or giantin by immunofluorescence. In A and B, the Sar1pdn stock concentration was 0.15 mg/ml and the micrographs show cells double labeled for GalNAcT2-GFP and giantin. Asterisks over the cell nuclei indicate microinjected cells. Noninjected control cells are unmarked. Note that at this concentration GalNAcT2 had redistributed into an ER distribution in all four microinjected cells shown. Giantin, however, often displayed a more mixed distribution; all microinjected cells exhibited at least a partial diffuse cytoplasmic distribution for giantin with the left-hand cell exhibiting a full redistribution of giantin, and the other three injected cells also showing some concentrated juxtanuclear staining indicative of residual Golgi apparatus. In C, the incidence of microinjected cells displaying an overall pronounced juxtanuclear distribution for GalNAcT2, giantin, or GM130 was scored relative to the Sar1pdn stock concentration as described previously (Girod et al., 1999). Two sets of data points are shown for GalNAcT2 because it was scored separately in giantin- and GM130-stained cells. In D, the incidence of microinjected cells displaying an overall pronounced juxtanuclear distribution for GalNAcT2 or giantin was scored relative to the pSARAdn stock concentration. In E and F, the pSARAdn stock was either 8 ng or 200 ng/μl. At the low pSARAdn concentration, a mixed pattern of giantin redistribution was common, whereas at the high concentration little incidence of juxtanuclear giantin staining was seen. The end giantin distribution was more granular than the continuous ER-like GalNAcT2 distribution. A coinjection marker was used to distinguish microinjected cells independently of phenotype. The average diameter of a HeLa nucleus is 10 μm.

Figure 2.
Figure 2.

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.

Figure 3.
Figure 3.

Golgi apparatus–specific O-glycosylation occurs within the ER of Sar1pdn-microinjected cells. Cells (asterisks) were microinjected with Sar1pdn and at the end of 6 h, fixed and stained with rhodamine-conjugated Helix pomatia lectin. The target cell population stably expresses an artificial ER-localized O-glycosylation substrate, CD8, fused with the ER retention domain of glucuronyltransferase. Helix pomatia lectin is a lectin specific for O-linked terminal α-GalNAc. In the microinjected cells, distinct staining of the nuclear envelope and into the cytoplasm was apparent. In the noninjected cells (arrowheads), a distinct and fainter concentration of staining was apparent in the juxtanuclear Golgi complex–like region.

Figure 4.
Figure 4.

Rapid redistribution of p27 in cells expressing pSARAdn. HeLa cells stably expressing GalNAcT2-GFP were microinjected with pSARAdn (A and B). At the end of a 2 h pSARAdn expression period, cells were fixed and stained for p27 using a Cy3-conjugated second antibody. p27 (A) appeared to lose juxtanuclear Golgi apparatus–like localization faster than GalNAcT2 (B) in the same cells. Wild-type HeLa cells were microinjected with Sar1pdn in the presence of CHX and incubated for 6 h postinjection (C). In these wild-type cells, p27 had completely lost juxtanuclear Golgi apparatus–like localization, and in favorable cells p27 staining appeared to localize to the nuclear envelope, e.g., arrowhead, inset. Asterisks indicate microinjected cells.

Figure 5.
Figure 5.

Similar redistribution kinetics for giantin and GalNAcT2-GFP in cells expressing pSARAdn. HeLa cells stably transfected with GalNAcT2-GFP were microinjected with pSARAdn in the absence of CHX. After 1 h (A and B), 2 h (C and D), or 6 h (E and F) expression period, cells were fixed and stained for giantin using a Cy3-conjugated second antibody. Giantin appeared to lose juxtanuclear Golgi apparatus–like localization with the same kinetics as GalNAcT2. As shown in 3.7× higher magnification inset to F, redistributed giantin and GalNAcT2-GFP similarly rimmed the nuclear envelope. As shown in the inset to E, giantin similarly redistributed in wild-type HeLa cells microinjected with Sar1pdn in the presence of CHX. Asterisks indicate microinjected cells.

Figure 6.
Figure 6.

Quantification and demonstration of the appearance of a series of Golgi apparatus cisternal and TGN proteins in the ER. In A, HeLa cells stably overexpressing a series of cisternal-to-TGN proteins were microinjected with Sar1pdn in the presence of CHX and appearance in the ER quantified as described previously (Girod et al., 1999). In B–D, TGN46 (B), GalNAcT2 (C), and GalT (D) in wild-type HeLa cells microinjected with 1.7 mg/ml Sar1pdn in the presence of CHX were localized 6 h post-injection. Asterisks indicate microinjected cells.

Figure 7.
Figure 7.

Kinetics of GalNAcT2-GFP transfer between the Golgi apparatus and ER from photobleaching (A) or quantification of Golgi pixel mass (B). In A, fluorescence intensity over the ER region of individual cells was quantified before and after a photobleach in the presence of the protein synthesis inhibitor CHX. In this experiment, there is no ER exit block. The irregular line function of the ZEISS LSM510 microscope software was used to encircle the ER and the ER was then photobleached. Inset, qualitative evidence that the bleach is selective for the ER. The data are for three cells and the average intensity and standard deviation are shown. About 35% of the ER-localized GFP was photobleached in each case. A partial photobleach rather than complete was chosen to minimize possible photodamage. Quantitatively, recovery of ER fluorescence was best fit by a single exponential function and had a half-time of 15 min. To image the less intense ER contribution, the Golgi apparatus was imaged to oversaturation. In B, loss of fluorescence from the Golgi apparatus following Sar1pdn microinjection in the presence of CHX was analyzed based on mass action kinetics. Golgi area was scored by using the irregular line tool of the NIH Image program to enclose the juxtanuclear concentrated GalNAcT2-GFP pixels in ∼10 cells/time point. The total pixel number within the enclosed area was then quantified for 10 cells. Pixel number was used rather than summed fluorescence intensity because the Golgi apparatus had been imaged to oversaturation in the micrograph set in order to image the less intense ER contribution (see Fig. 6). The decrease in Golgi pixels with fluorescent signal was described well by a first-order rate constant (KGolgi-ER = −0.5703 h−1; R = 0.99). The half-time for depletion is 1 h. Fluorescence appearance in the ER is described well by a first-order process with no depletion term, indicating that ER exit in the presence of Sar1p is not significant (unpublished data). The error bars in B indicate standard error of the mean.

Figure 8.
Figure 8.

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.

Figure 9.
Figure 9.

p115 loses its juxtanuclear Golgi region localization in a similar manner to GalNAcT2-GFP in pSARAdn-expressing cells. GalNAcT2-GFP HeLa cells were microinjected with pSARAdn (asterisks) and fixed at 0 t and 1, 3, and 6 h. Cells were stained for p115 using a Cy3 second antibody. Only the 0 t and 3 h time points are shown.

Figure 10.
Figure 10.

GM130 loses its juxtanuclear Golgi region localization in a similar manner to GalNAcT2-GFP in pSARAdn-expressing cells. GalNAcT2-GFP HeLa cells were microinjected with pSARAdn (asterisks) and fixed at 2, 4, and 6 h. Cells were stained for GM130 using a Cy3 second antibody.

Figure 11.
Figure 11.

GM130 has a mixed diffuse and granular distribution in HeLa cells microinjected with pSARAdn and fixed with methanol. HeLa cells stably expressing GalNAcT2-VSV were microinjected with 150 ng/μl stock concentration pSARAdn, incubated for 6 h, and fixed with −20°C methanol to give better preservation of microtubules than with formaldehyde fixation. Microinjected cells were identified by the ER distribution of GalNAcT2 after antibody staining. GM130 has a diffuse to granular distribution in the microinjected cells. A, GM130; B, GalNAcT2. Asterisks indicate microinjected cells.

Figure 12.
Figure 12.

Sar1pdn blocks the juxtanuclear accumulation of Golgi region proteins in a BFA washout protocol. HeLa cells stably expressing GalNAcT2-GFP were exposed to BFA for 30 min in the presence of CHX, fixed, and then stained. A, GalNAcT2-GFP; B, giantin in same cells, double labeling; C, GM130. HeLa cells stably expressing GalNAcT2-GFP were microinjected with either 0.72 or 2.85 mg/ml Sar1pdn, incubated with BFA for 30 min in the presence of CHX, and then incubated for an additional 2 h in the presence of CHX after BFA washout. D, GalNAcT2-GFP, 0.72 mg/ml Sar1pdn; E, giantin, 2.85 mg/ml Sar1pdn; F, GM130, double labeling of same cells shown in D; G, p27, 2.85 mg/ml Sar1pdn; H, quantification of fluorescence distributions after BFA washout, scoring as described previously by Girod et al. (1999). Asterisks indicate microinjected cells.

Comment in

  • Let's make Golgi.

    Wells WA. Wells WA. J Cell Biol. 2001 Nov 12;155(4):498-9. doi: 10.1083/jcb.200110112. Epub 2001 Nov 12. J Cell Biol. 2001. PMID: 11706045 Free PMC article.

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