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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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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.
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fig7: 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.

Mentions: The above experiments indicate eight different cis-to-TGN integral Golgi apparatus membrane proteins plus GM130 and giantin cycle in response to an ER exit block. To provide direct evidence that such recycling is not an artifact of the ER exit block, the equilibration of GalNAcT2-GFP between two pools, the Golgi apparatus and ER, was characterized by a photobleaching approach. As shown in Fig. 7 A, inset, GalNAcT2-GFP fluorescence in cells stably expressing high levels of the protein can be detected at high intensities in association with the juxtanuclear Golgi apparatus and at low intensities over the cytoplasm in association with the ER. The ER fluorescence was not due to newly synthesized GalNAcT2-GFP as it did not chase out of the ER with incubation of the cells in the presence of CHX (unpublished data; no ER exit block). To test whether the two GalNAcT2-GFP pools were in equilibrium with one another, GalNAcT2-GFP fluorescence over the cytoplasm was photobleached in the presence of CHX and the same cell was then imaged at 10 min intervals in the presence of drug. As shown qualitatively in Fig. 7 A, inset, complete photorecovery was seen. Quantitatively, recovery had a half time of ∼15 min, indicating that indeed GalNAcT2-GFP normally is transported in a fairly rapid manner between the Golgi apparatus and ER (Fig. 7 A). This half time was fourfold faster than that for the loss of GalNAcT2-GFP–marked Golgi matrix mass (average Golgi matrix pixel number per cells) in response to an ER exit block (t1/2 = 1 h; Fig. 7 B). The kinetics for FRAP are similar to those reported by Zaal et al. (1999). As an additional control that GalNAcT2-GFP had accumulated in the ER, a continuous structure, in response to the ER exit block, HeLa cells following a 6 h pSARAdn expression period were photobleached over approximately one-half the cell area and imaged at 1 min intervals. In these cells, the Golgi apparatus had redistributed, presumably to the ER. Extensive photorecovery was seen over a 4 min time period over the bleached area (unpublished data), the expected result for an interconnected ER network.


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)

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.
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fig7: 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.
Mentions: The above experiments indicate eight different cis-to-TGN integral Golgi apparatus membrane proteins plus GM130 and giantin cycle in response to an ER exit block. To provide direct evidence that such recycling is not an artifact of the ER exit block, the equilibration of GalNAcT2-GFP between two pools, the Golgi apparatus and ER, was characterized by a photobleaching approach. As shown in Fig. 7 A, inset, GalNAcT2-GFP fluorescence in cells stably expressing high levels of the protein can be detected at high intensities in association with the juxtanuclear Golgi apparatus and at low intensities over the cytoplasm in association with the ER. The ER fluorescence was not due to newly synthesized GalNAcT2-GFP as it did not chase out of the ER with incubation of the cells in the presence of CHX (unpublished data; no ER exit block). To test whether the two GalNAcT2-GFP pools were in equilibrium with one another, GalNAcT2-GFP fluorescence over the cytoplasm was photobleached in the presence of CHX and the same cell was then imaged at 10 min intervals in the presence of drug. As shown qualitatively in Fig. 7 A, inset, complete photorecovery was seen. Quantitatively, recovery had a half time of ∼15 min, indicating that indeed GalNAcT2-GFP normally is transported in a fairly rapid manner between the Golgi apparatus and ER (Fig. 7 A). This half time was fourfold faster than that for the loss of GalNAcT2-GFP–marked Golgi matrix mass (average Golgi matrix pixel number per cells) in response to an ER exit block (t1/2 = 1 h; Fig. 7 B). The kinetics for FRAP are similar to those reported by Zaal et al. (1999). As an additional control that GalNAcT2-GFP had accumulated in the ER, a continuous structure, in response to the ER exit block, HeLa cells following a 6 h pSARAdn expression period were photobleached over approximately one-half the cell area and imaged at 1 min intervals. In these cells, the Golgi apparatus had redistributed, presumably to the ER. Extensive photorecovery was seen over a 4 min time period over the bleached area (unpublished data), the expected result for an interconnected ER network.

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