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Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Mironov AA, Beznoussenko GV, Nicoziani P, Martella O, Trucco A, Kweon HS, Di Giandomenico D, Polishchuk RS, Fusella A, Lupetti P, Berger EG, Geerts WJ, Koster AJ, Burger KN, Luini A - J. Cell Biol. (2001)

Bottom Line: Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae.Transport was followed using a combination of video and EM, providing high resolution in time and space.Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," 66030 Santa Maria Imbaro, Chieti, Italy.

ABSTRACT
Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

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PC-I and VSVG are transported through the Golgi complex at indistinguishable rates. Human fibroblasts were subjected to different synchronization protocols (see below), fixed at the times indicated in the figure after release of the temperature block, and then double immunolabeled for PC-I and VSVG. For the sake of space, in most panels the two colors are presented only in the merged form. (A–I) Large-pulse protocol. (L–N) Small-pulse protocol. The inset in panel I shows labeling for VSVG on the membrane surface (labeled without permeabilization with an antibody against the lumenal epitope). The colocalization between PC-I and VSVG was significant at all time points. (O–R) Quantification and time course of the passage of VSVG and through the Golgi under the large-, small-, and intermediate-pulse, and exiting-wave protocols. The values were obtained by measuring the average fluorescence intensities (in arbitrary units) of each cargo in the Golgi area and in the nuclear envelope (ER, with average fluorescence of 1). The differences between the Golgi and the ER values for each cargo were then calculated and plotted as a function of time. Clearly, this difference (Golgi minus ER) increases as cargoes exit the ER and enter the Golgi, and then it decreases when the cargoes exit the Golgi for the plasma membrane. The average transit time of the cargoes through the Golgi area is defined as the lag (indicated by the discontinuous line in O and Q) between the half time of the rising phase and that of the decay phase. The transit time was ∼20 min for the large-pulse, and 8–10 min for the small- and intermediate-pulse protocols. The cargo clearance time from the Golgi under the exiting-wave protocol is defined as the lag between the start of the 40°C block and the half time of the decay phase of the curve (R); it was ∼7–8 min. It is apparent that VSVG and PC-I move simultaneously through the Golgi area under all the protocols, whereas the rate of traffic differs between protocols. Each value represents the average of 7–15 independent measurements from at least three different experiments. The SDs did not exceed 15% of the mean. Bar, 10 μm.
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fig2: PC-I and VSVG are transported through the Golgi complex at indistinguishable rates. Human fibroblasts were subjected to different synchronization protocols (see below), fixed at the times indicated in the figure after release of the temperature block, and then double immunolabeled for PC-I and VSVG. For the sake of space, in most panels the two colors are presented only in the merged form. (A–I) Large-pulse protocol. (L–N) Small-pulse protocol. The inset in panel I shows labeling for VSVG on the membrane surface (labeled without permeabilization with an antibody against the lumenal epitope). The colocalization between PC-I and VSVG was significant at all time points. (O–R) Quantification and time course of the passage of VSVG and through the Golgi under the large-, small-, and intermediate-pulse, and exiting-wave protocols. The values were obtained by measuring the average fluorescence intensities (in arbitrary units) of each cargo in the Golgi area and in the nuclear envelope (ER, with average fluorescence of 1). The differences between the Golgi and the ER values for each cargo were then calculated and plotted as a function of time. Clearly, this difference (Golgi minus ER) increases as cargoes exit the ER and enter the Golgi, and then it decreases when the cargoes exit the Golgi for the plasma membrane. The average transit time of the cargoes through the Golgi area is defined as the lag (indicated by the discontinuous line in O and Q) between the half time of the rising phase and that of the decay phase. The transit time was ∼20 min for the large-pulse, and 8–10 min for the small- and intermediate-pulse protocols. The cargo clearance time from the Golgi under the exiting-wave protocol is defined as the lag between the start of the 40°C block and the half time of the decay phase of the curve (R); it was ∼7–8 min. It is apparent that VSVG and PC-I move simultaneously through the Golgi area under all the protocols, whereas the rate of traffic differs between protocols. Each value represents the average of 7–15 independent measurements from at least three different experiments. The SDs did not exceed 15% of the mean. Bar, 10 μm.

Mentions: First, we used the large-pulse protocol, which results in a large buildup of PC-I and VSVG in the IC. Fig. 2 shows by immunofluorescence that when the cells were shifted from 40 to 15°C, both cargoes exited the ER and, after 2 h, were present in numerous bright spots (IC elements) distributed throughout the cytoplasm. PC-I and VSVG exhibited a good degree of overlap in these structures. When the temperature was shifted back to 40°C, the distribution of both proteins in the IC began to change 3–4 min later: PC-I and VSVG came closer to the Golgi, and at 4–5 min they had reached the Golgi area (identified by the bona fide Golgi marker giantin, unpublished data) where they resided for an average of 20 min (transit time, legend to Fig. 2). Finally, both cargoes exited the Golgi together in distinct spots, probably representing post-Golgi carriers, in which they colocalized. (Fig. 2, A–I). Thus, the rates of passage of PC-I and VSVG through the secretory system were indistinguishable.


Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Mironov AA, Beznoussenko GV, Nicoziani P, Martella O, Trucco A, Kweon HS, Di Giandomenico D, Polishchuk RS, Fusella A, Lupetti P, Berger EG, Geerts WJ, Koster AJ, Burger KN, Luini A - J. Cell Biol. (2001)

PC-I and VSVG are transported through the Golgi complex at indistinguishable rates. Human fibroblasts were subjected to different synchronization protocols (see below), fixed at the times indicated in the figure after release of the temperature block, and then double immunolabeled for PC-I and VSVG. For the sake of space, in most panels the two colors are presented only in the merged form. (A–I) Large-pulse protocol. (L–N) Small-pulse protocol. The inset in panel I shows labeling for VSVG on the membrane surface (labeled without permeabilization with an antibody against the lumenal epitope). The colocalization between PC-I and VSVG was significant at all time points. (O–R) Quantification and time course of the passage of VSVG and through the Golgi under the large-, small-, and intermediate-pulse, and exiting-wave protocols. The values were obtained by measuring the average fluorescence intensities (in arbitrary units) of each cargo in the Golgi area and in the nuclear envelope (ER, with average fluorescence of 1). The differences between the Golgi and the ER values for each cargo were then calculated and plotted as a function of time. Clearly, this difference (Golgi minus ER) increases as cargoes exit the ER and enter the Golgi, and then it decreases when the cargoes exit the Golgi for the plasma membrane. The average transit time of the cargoes through the Golgi area is defined as the lag (indicated by the discontinuous line in O and Q) between the half time of the rising phase and that of the decay phase. The transit time was ∼20 min for the large-pulse, and 8–10 min for the small- and intermediate-pulse protocols. The cargo clearance time from the Golgi under the exiting-wave protocol is defined as the lag between the start of the 40°C block and the half time of the decay phase of the curve (R); it was ∼7–8 min. It is apparent that VSVG and PC-I move simultaneously through the Golgi area under all the protocols, whereas the rate of traffic differs between protocols. Each value represents the average of 7–15 independent measurements from at least three different experiments. The SDs did not exceed 15% of the mean. Bar, 10 μm.
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fig2: PC-I and VSVG are transported through the Golgi complex at indistinguishable rates. Human fibroblasts were subjected to different synchronization protocols (see below), fixed at the times indicated in the figure after release of the temperature block, and then double immunolabeled for PC-I and VSVG. For the sake of space, in most panels the two colors are presented only in the merged form. (A–I) Large-pulse protocol. (L–N) Small-pulse protocol. The inset in panel I shows labeling for VSVG on the membrane surface (labeled without permeabilization with an antibody against the lumenal epitope). The colocalization between PC-I and VSVG was significant at all time points. (O–R) Quantification and time course of the passage of VSVG and through the Golgi under the large-, small-, and intermediate-pulse, and exiting-wave protocols. The values were obtained by measuring the average fluorescence intensities (in arbitrary units) of each cargo in the Golgi area and in the nuclear envelope (ER, with average fluorescence of 1). The differences between the Golgi and the ER values for each cargo were then calculated and plotted as a function of time. Clearly, this difference (Golgi minus ER) increases as cargoes exit the ER and enter the Golgi, and then it decreases when the cargoes exit the Golgi for the plasma membrane. The average transit time of the cargoes through the Golgi area is defined as the lag (indicated by the discontinuous line in O and Q) between the half time of the rising phase and that of the decay phase. The transit time was ∼20 min for the large-pulse, and 8–10 min for the small- and intermediate-pulse protocols. The cargo clearance time from the Golgi under the exiting-wave protocol is defined as the lag between the start of the 40°C block and the half time of the decay phase of the curve (R); it was ∼7–8 min. It is apparent that VSVG and PC-I move simultaneously through the Golgi area under all the protocols, whereas the rate of traffic differs between protocols. Each value represents the average of 7–15 independent measurements from at least three different experiments. The SDs did not exceed 15% of the mean. Bar, 10 μm.
Mentions: First, we used the large-pulse protocol, which results in a large buildup of PC-I and VSVG in the IC. Fig. 2 shows by immunofluorescence that when the cells were shifted from 40 to 15°C, both cargoes exited the ER and, after 2 h, were present in numerous bright spots (IC elements) distributed throughout the cytoplasm. PC-I and VSVG exhibited a good degree of overlap in these structures. When the temperature was shifted back to 40°C, the distribution of both proteins in the IC began to change 3–4 min later: PC-I and VSVG came closer to the Golgi, and at 4–5 min they had reached the Golgi area (identified by the bona fide Golgi marker giantin, unpublished data) where they resided for an average of 20 min (transit time, legend to Fig. 2). Finally, both cargoes exited the Golgi together in distinct spots, probably representing post-Golgi carriers, in which they colocalized. (Fig. 2, A–I). Thus, the rates of passage of PC-I and VSVG through the secretory system were indistinguishable.

Bottom Line: Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae.Transport was followed using a combination of video and EM, providing high resolution in time and space.Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," 66030 Santa Maria Imbaro, Chieti, Italy.

ABSTRACT
Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

Show MeSH
Related in: MedlinePlus