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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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VSVG and ssHRP are excluded from Golgi vesicles during transit through the Golgi complex. Human fibroblasts (a–c) and COS-7 cells (d–f) were subjected to the ER accumulation–chase protocol (D) or the large (C) or the intermediate (A and B) pulse protocols, fixed 7 min after the release of the temperature block (A–D), and then processed for labeling. In the experiments in (A) and (B) they were labeled for VSVG by the cryo-immunogold technique, in C by the nanogold gold enhance technique, and in D (which shows a tangential thick section), by the preembedding immunoperoxidase method. Also in A, the GM130 protein is labeled (small particles). (e and f) For ssHRP experiments, COS-7 cells were transfected with ssHRP, fixed at steady state, and then processed for detection of HRP. (E) Perpendicular and (F) tangential section. Irrespective of the labeling and sectioning technique, the round (vesicular) profiles (arrows) are almost always devoid of cargo, whereas cisternae are labeled. Bar: (A, D, and E) 110 nm; (B) 120 nm; (C) 90 nm; (F) 200 nm.
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fig7: VSVG and ssHRP are excluded from Golgi vesicles during transit through the Golgi complex. Human fibroblasts (a–c) and COS-7 cells (d–f) were subjected to the ER accumulation–chase protocol (D) or the large (C) or the intermediate (A and B) pulse protocols, fixed 7 min after the release of the temperature block (A–D), and then processed for labeling. In the experiments in (A) and (B) they were labeled for VSVG by the cryo-immunogold technique, in C by the nanogold gold enhance technique, and in D (which shows a tangential thick section), by the preembedding immunoperoxidase method. Also in A, the GM130 protein is labeled (small particles). (e and f) For ssHRP experiments, COS-7 cells were transfected with ssHRP, fixed at steady state, and then processed for detection of HRP. (E) Perpendicular and (F) tangential section. Irrespective of the labeling and sectioning technique, the round (vesicular) profiles (arrows) are almost always devoid of cargo, whereas cisternae are labeled. Bar: (A, D, and E) 110 nm; (B) 120 nm; (C) 90 nm; (F) 200 nm.

Mentions: The second question in this section is whether VSVG is excluded from Golgi vesicles during intra-Golgi traffic. Although VSVG has been found in COPI vesicles at concentrations comparable to those in cisternae (Orci et al., 1986, 1993) in in vitro Golgi preparations, to our knowledge, analogous data in living cells have not been reported thus far. For this analysis we used not only the small-pulse (Fig. 5), but also the large-pulse and the ER accumulation–chase protocols, which generate higher fluxes of membrane and cargo through the Golgi. Cells (both fibroblasts and COS-7 cells) were fixed 8–9 min after release of the temperature block, i.e., when the flux through the stack is high. The analysis was limited to the vesicular profiles adjacent (lateral) to labeled cisternae (see Materials and methods). We assume that these profiles represent bona fide vesicles based on published evidence, including EM tomography data by Marsh et al. (2001) indicating that vesicles represent a major fraction of the round profiles in the vicinity of cisternae, and data of Orci et al. (1997), showing that most of these profiles are COPI coated. These vesicles can in principle derive from Golgi cisternae or from neighboring IC elements. To distinguish their origin, we labeled them for the IC marker GM130 (Marra et al., 2001). None of the vesicles was labeled, ruling out the IC as their source and indicating that they derive from the Golgi stack. Cells were then labeled for VSVG to determine its level in the Golgi vesicles. Several labeling methods were used to avoid systematic artifacts. First, we used the cryoimmunogold technique and an antibody against a lumenal VSVG epitope. The Golgi stacks were abundantly labeled, whereas only a few gold particles were found in the neighboring vesicular profiles (Fig. 7, A and B). Morphometry showed that the density of particles was sixfold lower in vesicular profiles than in adjacent cisternae (Table I). The density ratio between the two structures was not affected by the level of expression of VSVG, which varied up to threefold between different cells (unpublished data). To rule out the possibility that the lumenal VSVG antigen in vesicles might be masked by lumenal proteins or might be less accessible than in cisternae because of geometry factors, we next used an antibody against a cytosolic VSVG epitope. The ratio between cisternal and vesicular labeling densities remained close to six (unpublished data). The same experiment (using the cytosolic epitope) was then repeated using the preembedding nanogold gold enhance technique, in which the problems of epitope accessibility are different than those encountered in cryogold labeling. Under these conditions, the ratio was close to eight (Fig. 7 C; Table I). The preembedding immunoperoxidase technique using an antibody against the lumenal VSVG epitope was also applied. The results in panel 7 D (tangential semithick section of a cisterna) again show intense labeling in cisternae but no labeling in vesicular profiles. Finally, we used another antigen and a radically different approach. Cells were transfected with secretory soluble HRP (ssHRP) (Connolly et al., 1994), whose detection is free of all the problems related to the access of antibody to epitope, and fixed at steady state (ssHRP cannot be synchronized). Cells were fixed and subjected to the HRP reaction procedure. Once again, vesicular profiles were depleted of cargo, i.e., unstained, whereas most of the cisternae were stained, often intensely (Fig. 7, E and F), indicating that secretory HRP is much less concentrated in vesicles than in cisternae. In summary, by using a variety of labeling techniques, we find that small diffusible secretory proteins are depleted in Golgi vesicles as compared with the stack.


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)

VSVG and ssHRP are excluded from Golgi vesicles during transit through the Golgi complex. Human fibroblasts (a–c) and COS-7 cells (d–f) were subjected to the ER accumulation–chase protocol (D) or the large (C) or the intermediate (A and B) pulse protocols, fixed 7 min after the release of the temperature block (A–D), and then processed for labeling. In the experiments in (A) and (B) they were labeled for VSVG by the cryo-immunogold technique, in C by the nanogold gold enhance technique, and in D (which shows a tangential thick section), by the preembedding immunoperoxidase method. Also in A, the GM130 protein is labeled (small particles). (e and f) For ssHRP experiments, COS-7 cells were transfected with ssHRP, fixed at steady state, and then processed for detection of HRP. (E) Perpendicular and (F) tangential section. Irrespective of the labeling and sectioning technique, the round (vesicular) profiles (arrows) are almost always devoid of cargo, whereas cisternae are labeled. Bar: (A, D, and E) 110 nm; (B) 120 nm; (C) 90 nm; (F) 200 nm.
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fig7: VSVG and ssHRP are excluded from Golgi vesicles during transit through the Golgi complex. Human fibroblasts (a–c) and COS-7 cells (d–f) were subjected to the ER accumulation–chase protocol (D) or the large (C) or the intermediate (A and B) pulse protocols, fixed 7 min after the release of the temperature block (A–D), and then processed for labeling. In the experiments in (A) and (B) they were labeled for VSVG by the cryo-immunogold technique, in C by the nanogold gold enhance technique, and in D (which shows a tangential thick section), by the preembedding immunoperoxidase method. Also in A, the GM130 protein is labeled (small particles). (e and f) For ssHRP experiments, COS-7 cells were transfected with ssHRP, fixed at steady state, and then processed for detection of HRP. (E) Perpendicular and (F) tangential section. Irrespective of the labeling and sectioning technique, the round (vesicular) profiles (arrows) are almost always devoid of cargo, whereas cisternae are labeled. Bar: (A, D, and E) 110 nm; (B) 120 nm; (C) 90 nm; (F) 200 nm.
Mentions: The second question in this section is whether VSVG is excluded from Golgi vesicles during intra-Golgi traffic. Although VSVG has been found in COPI vesicles at concentrations comparable to those in cisternae (Orci et al., 1986, 1993) in in vitro Golgi preparations, to our knowledge, analogous data in living cells have not been reported thus far. For this analysis we used not only the small-pulse (Fig. 5), but also the large-pulse and the ER accumulation–chase protocols, which generate higher fluxes of membrane and cargo through the Golgi. Cells (both fibroblasts and COS-7 cells) were fixed 8–9 min after release of the temperature block, i.e., when the flux through the stack is high. The analysis was limited to the vesicular profiles adjacent (lateral) to labeled cisternae (see Materials and methods). We assume that these profiles represent bona fide vesicles based on published evidence, including EM tomography data by Marsh et al. (2001) indicating that vesicles represent a major fraction of the round profiles in the vicinity of cisternae, and data of Orci et al. (1997), showing that most of these profiles are COPI coated. These vesicles can in principle derive from Golgi cisternae or from neighboring IC elements. To distinguish their origin, we labeled them for the IC marker GM130 (Marra et al., 2001). None of the vesicles was labeled, ruling out the IC as their source and indicating that they derive from the Golgi stack. Cells were then labeled for VSVG to determine its level in the Golgi vesicles. Several labeling methods were used to avoid systematic artifacts. First, we used the cryoimmunogold technique and an antibody against a lumenal VSVG epitope. The Golgi stacks were abundantly labeled, whereas only a few gold particles were found in the neighboring vesicular profiles (Fig. 7, A and B). Morphometry showed that the density of particles was sixfold lower in vesicular profiles than in adjacent cisternae (Table I). The density ratio between the two structures was not affected by the level of expression of VSVG, which varied up to threefold between different cells (unpublished data). To rule out the possibility that the lumenal VSVG antigen in vesicles might be masked by lumenal proteins or might be less accessible than in cisternae because of geometry factors, we next used an antibody against a cytosolic VSVG epitope. The ratio between cisternal and vesicular labeling densities remained close to six (unpublished data). The same experiment (using the cytosolic epitope) was then repeated using the preembedding nanogold gold enhance technique, in which the problems of epitope accessibility are different than those encountered in cryogold labeling. Under these conditions, the ratio was close to eight (Fig. 7 C; Table I). The preembedding immunoperoxidase technique using an antibody against the lumenal VSVG epitope was also applied. The results in panel 7 D (tangential semithick section of a cisterna) again show intense labeling in cisternae but no labeling in vesicular profiles. Finally, we used another antigen and a radically different approach. Cells were transfected with secretory soluble HRP (ssHRP) (Connolly et al., 1994), whose detection is free of all the problems related to the access of antibody to epitope, and fixed at steady state (ssHRP cannot be synchronized). Cells were fixed and subjected to the HRP reaction procedure. Once again, vesicular profiles were depleted of cargo, i.e., unstained, whereas most of the cisternae were stained, often intensely (Fig. 7, E and F), indicating that secretory HRP is much less concentrated in vesicles than in cisternae. In summary, by using a variety of labeling techniques, we find that small diffusible secretory proteins are depleted in Golgi vesicles as compared with the stack.

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