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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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Human fibroblasts can express and transport both PC-I aggregates and VSVG through the Golgi complex. Human fibroblasts were stimulated to synthesize PC-I and infected with ts045-VSV. After accumulation of both and PC-I and VSVG in the ER at 40°C for 3 h, cells were shifted to 32°C for 9 min and then fixed, permeabilized with saponin, and prepared for immunofluorescence or fixed and prepared for immuno-EM. (A and B) Immunofluorescent double labeling for VSVG (red) and PC-I (green). Both cargoes localize mostly in the Golgi area. The two labeling patterns are very similar. (C) Immuno-EM labeling of PC-I by the preembedding nanogold gold enhancement technique. An aggregate appears as thick cluster of gold particles in a tangential section of a Golgi cisterna. (D) Immuno-EM labeling of VSVG by the same technique. VSVG is distributed throughout the Golgi membranes, including PC-I–containing distensions. Many typical cisternal distensions (*) are seen within the Golgi ribbon (arrows). (E–H) Synchronization protocols. (E) Pulse protocols: cells were kept at 32°C in the presence of AA (50 μg/ml) for 3 h, shifted to 40°C for 3 h (in some experiments 1–2 h), and then shifted to 15°C for 2 h (large pulse), 45 min (intermediate pulse), or 15 min (small pulse), and finally shifted back to 40°C. (F) ER accumulation–chase: cells were kept at 40°C for 3 h in the absence of AA, and then shifted to 32°C in the presence of AA. (G) ER accumulation–pulse: same as for ER accumulation–chase except that cells were shifted back to 40°C after 5 min at 32°C. (H) Exiting wave protocol (Results). Bar: (A and B) 200 nm; (C and D) 2 μm.
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fig1: Human fibroblasts can express and transport both PC-I aggregates and VSVG through the Golgi complex. Human fibroblasts were stimulated to synthesize PC-I and infected with ts045-VSV. After accumulation of both and PC-I and VSVG in the ER at 40°C for 3 h, cells were shifted to 32°C for 9 min and then fixed, permeabilized with saponin, and prepared for immunofluorescence or fixed and prepared for immuno-EM. (A and B) Immunofluorescent double labeling for VSVG (red) and PC-I (green). Both cargoes localize mostly in the Golgi area. The two labeling patterns are very similar. (C) Immuno-EM labeling of PC-I by the preembedding nanogold gold enhancement technique. An aggregate appears as thick cluster of gold particles in a tangential section of a Golgi cisterna. (D) Immuno-EM labeling of VSVG by the same technique. VSVG is distributed throughout the Golgi membranes, including PC-I–containing distensions. Many typical cisternal distensions (*) are seen within the Golgi ribbon (arrows). (E–H) Synchronization protocols. (E) Pulse protocols: cells were kept at 32°C in the presence of AA (50 μg/ml) for 3 h, shifted to 40°C for 3 h (in some experiments 1–2 h), and then shifted to 15°C for 2 h (large pulse), 45 min (intermediate pulse), or 15 min (small pulse), and finally shifted back to 40°C. (F) ER accumulation–chase: cells were kept at 40°C for 3 h in the absence of AA, and then shifted to 32°C in the presence of AA. (G) ER accumulation–pulse: same as for ER accumulation–chase except that cells were shifted back to 40°C after 5 min at 32°C. (H) Exiting wave protocol (Results). Bar: (A and B) 200 nm; (C and D) 2 μm.

Mentions: Human fibroblasts are flat and suitable for morphological transport assays. When kept in growth medium (10% FCS) they synthesize PC-I in low amounts, but can be switched into a more differentiated PC-I–producing state by replacing 10% fetal calf serum with 1% adult calf serum for 3–10 h. Under the latter conditions, their rate of PC-I secretion is comparable to that of other “professional” PC-I secretors (Bonfanti et al., 1998). When infected with VSV at low concentrations for short incubation times (see Materials and methods), they express measurable amounts of the VSVG without significantly losing expression of PC-I (Fig. 1, A–D). They can also be transfected with VSVG or VSVG–GFP by electroporation. Thus, they are suitable for studies of dual PC-I and VSVG cargo transport.


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)

Human fibroblasts can express and transport both PC-I aggregates and VSVG through the Golgi complex. Human fibroblasts were stimulated to synthesize PC-I and infected with ts045-VSV. After accumulation of both and PC-I and VSVG in the ER at 40°C for 3 h, cells were shifted to 32°C for 9 min and then fixed, permeabilized with saponin, and prepared for immunofluorescence or fixed and prepared for immuno-EM. (A and B) Immunofluorescent double labeling for VSVG (red) and PC-I (green). Both cargoes localize mostly in the Golgi area. The two labeling patterns are very similar. (C) Immuno-EM labeling of PC-I by the preembedding nanogold gold enhancement technique. An aggregate appears as thick cluster of gold particles in a tangential section of a Golgi cisterna. (D) Immuno-EM labeling of VSVG by the same technique. VSVG is distributed throughout the Golgi membranes, including PC-I–containing distensions. Many typical cisternal distensions (*) are seen within the Golgi ribbon (arrows). (E–H) Synchronization protocols. (E) Pulse protocols: cells were kept at 32°C in the presence of AA (50 μg/ml) for 3 h, shifted to 40°C for 3 h (in some experiments 1–2 h), and then shifted to 15°C for 2 h (large pulse), 45 min (intermediate pulse), or 15 min (small pulse), and finally shifted back to 40°C. (F) ER accumulation–chase: cells were kept at 40°C for 3 h in the absence of AA, and then shifted to 32°C in the presence of AA. (G) ER accumulation–pulse: same as for ER accumulation–chase except that cells were shifted back to 40°C after 5 min at 32°C. (H) Exiting wave protocol (Results). Bar: (A and B) 200 nm; (C and D) 2 μm.
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Related In: Results  -  Collection

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fig1: Human fibroblasts can express and transport both PC-I aggregates and VSVG through the Golgi complex. Human fibroblasts were stimulated to synthesize PC-I and infected with ts045-VSV. After accumulation of both and PC-I and VSVG in the ER at 40°C for 3 h, cells were shifted to 32°C for 9 min and then fixed, permeabilized with saponin, and prepared for immunofluorescence or fixed and prepared for immuno-EM. (A and B) Immunofluorescent double labeling for VSVG (red) and PC-I (green). Both cargoes localize mostly in the Golgi area. The two labeling patterns are very similar. (C) Immuno-EM labeling of PC-I by the preembedding nanogold gold enhancement technique. An aggregate appears as thick cluster of gold particles in a tangential section of a Golgi cisterna. (D) Immuno-EM labeling of VSVG by the same technique. VSVG is distributed throughout the Golgi membranes, including PC-I–containing distensions. Many typical cisternal distensions (*) are seen within the Golgi ribbon (arrows). (E–H) Synchronization protocols. (E) Pulse protocols: cells were kept at 32°C in the presence of AA (50 μg/ml) for 3 h, shifted to 40°C for 3 h (in some experiments 1–2 h), and then shifted to 15°C for 2 h (large pulse), 45 min (intermediate pulse), or 15 min (small pulse), and finally shifted back to 40°C. (F) ER accumulation–chase: cells were kept at 40°C for 3 h in the absence of AA, and then shifted to 32°C in the presence of AA. (G) ER accumulation–pulse: same as for ER accumulation–chase except that cells were shifted back to 40°C after 5 min at 32°C. (H) Exiting wave protocol (Results). Bar: (A and B) 200 nm; (C and D) 2 μm.
Mentions: Human fibroblasts are flat and suitable for morphological transport assays. When kept in growth medium (10% FCS) they synthesize PC-I in low amounts, but can be switched into a more differentiated PC-I–producing state by replacing 10% fetal calf serum with 1% adult calf serum for 3–10 h. Under the latter conditions, their rate of PC-I secretion is comparable to that of other “professional” PC-I secretors (Bonfanti et al., 1998). When infected with VSV at low concentrations for short incubation times (see Materials and methods), they express measurable amounts of the VSVG without significantly losing expression of PC-I (Fig. 1, A–D). They can also be transfected with VSVG or VSVG–GFP by electroporation. Thus, they are suitable for studies of dual PC-I and VSVG cargo transport.

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