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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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Transit of PC-I aggregates through the Golgi stack. Human fibroblasts were subjected to the small-pulse protocol, fixed at the times indicated below after release of the 15°C block, and prepared for immunogold labeling for PC-I (10-nm particles in A–C, 5-nm particles in D), VSVG (10-nm particles in D) and the Golgi markers GM130, GT, and TGN46 (5-nm particles). 3 min after the shift (A), VSVG colocalized with GM130; 7 min after the shift, VSVG colocalized with GT (B) but not with GM130 (C). Note that GT is present in the membrane surrounding the aggregate (arrows). (D) PC and VSVG colocalize at one pole of the stack. Bar: (A and B) 80 nm; (C) 130 nm; (D) 60 nm.
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fig6: Transit of PC-I aggregates through the Golgi stack. Human fibroblasts were subjected to the small-pulse protocol, fixed at the times indicated below after release of the 15°C block, and prepared for immunogold labeling for PC-I (10-nm particles in A–C, 5-nm particles in D), VSVG (10-nm particles in D) and the Golgi markers GM130, GT, and TGN46 (5-nm particles). 3 min after the shift (A), VSVG colocalized with GM130; 7 min after the shift, VSVG colocalized with GT (B) but not with GM130 (C). Note that GT is present in the membrane surrounding the aggregate (arrows). (D) PC and VSVG colocalize at one pole of the stack. Bar: (A and B) 80 nm; (C) 130 nm; (D) 60 nm.

Mentions: Because the Golgi area contains diverse structures, we next analyzed the progression of the two cargoes through the main Golgi subcompartments, namely the cis-Golgi network (CGN), the stack, and the trans-Golgi network (TGN), by relating the localization of PC-I and VSVG to the known markers of these compartments, the Golgi matrix protein (GM)130, galactosyl-transferase (GT), and TGN46. This is possible by immunofluorescence microscopy in these cells, as can be seen in Fig. 3, A–C, most likely because the noncisternal components of the CGN and TGN extend sufficiently far from the side of the stack (as can be appreciated by inspecting their ultrastructure; unpublished data) to allow for partial resolution of these three compartments at the light microscopy level. The small-pulse protocol was again used for these experiments. Both cargoes reached the Golgi area 3–4 min after release of the block. At this time, their colocalization with GM130 was good, with GT poor, and with TGN-46 nearly absent, indicating that they resided in the CGN. During the next 2–3 min, the overlap of the cargoes with GM130 decreased and that with GT increased, indicating that cargo transfer into the stacks was taking place. PC-I and VSVG then remained in the GT zone until ∼10 min, and finally shifted toward the TGN (judging from colocalization with TGN-46) where they remained until released from the Golgi in post-TGN carriers (Fig. 3, D–N). Thus, three stages of traffic in the Golgi area can be distinguished, throughout which PC-I and VSVG colocalize nearly perfectly (see below) and transit together at the same rates. The degree of overlap of each of the two cargoes with the markers of the three Golgi subcompartments during passage through the Golgi can be quantified. The results (Fig. 3, O–Q) underscore once again the remarkable coupling between the movements of the two cargoes, and confirm with a high degree of precision that VSVG and PC-I move together through the transport pathway. Similar results were obtained, with higher spatial resolution, in immuno-EM experiments (see Figs. 5 and 6 and related text).


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)

Transit of PC-I aggregates through the Golgi stack. Human fibroblasts were subjected to the small-pulse protocol, fixed at the times indicated below after release of the 15°C block, and prepared for immunogold labeling for PC-I (10-nm particles in A–C, 5-nm particles in D), VSVG (10-nm particles in D) and the Golgi markers GM130, GT, and TGN46 (5-nm particles). 3 min after the shift (A), VSVG colocalized with GM130; 7 min after the shift, VSVG colocalized with GT (B) but not with GM130 (C). Note that GT is present in the membrane surrounding the aggregate (arrows). (D) PC and VSVG colocalize at one pole of the stack. Bar: (A and B) 80 nm; (C) 130 nm; (D) 60 nm.
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Related In: Results  -  Collection

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fig6: Transit of PC-I aggregates through the Golgi stack. Human fibroblasts were subjected to the small-pulse protocol, fixed at the times indicated below after release of the 15°C block, and prepared for immunogold labeling for PC-I (10-nm particles in A–C, 5-nm particles in D), VSVG (10-nm particles in D) and the Golgi markers GM130, GT, and TGN46 (5-nm particles). 3 min after the shift (A), VSVG colocalized with GM130; 7 min after the shift, VSVG colocalized with GT (B) but not with GM130 (C). Note that GT is present in the membrane surrounding the aggregate (arrows). (D) PC and VSVG colocalize at one pole of the stack. Bar: (A and B) 80 nm; (C) 130 nm; (D) 60 nm.
Mentions: Because the Golgi area contains diverse structures, we next analyzed the progression of the two cargoes through the main Golgi subcompartments, namely the cis-Golgi network (CGN), the stack, and the trans-Golgi network (TGN), by relating the localization of PC-I and VSVG to the known markers of these compartments, the Golgi matrix protein (GM)130, galactosyl-transferase (GT), and TGN46. This is possible by immunofluorescence microscopy in these cells, as can be seen in Fig. 3, A–C, most likely because the noncisternal components of the CGN and TGN extend sufficiently far from the side of the stack (as can be appreciated by inspecting their ultrastructure; unpublished data) to allow for partial resolution of these three compartments at the light microscopy level. The small-pulse protocol was again used for these experiments. Both cargoes reached the Golgi area 3–4 min after release of the block. At this time, their colocalization with GM130 was good, with GT poor, and with TGN-46 nearly absent, indicating that they resided in the CGN. During the next 2–3 min, the overlap of the cargoes with GM130 decreased and that with GT increased, indicating that cargo transfer into the stacks was taking place. PC-I and VSVG then remained in the GT zone until ∼10 min, and finally shifted toward the TGN (judging from colocalization with TGN-46) where they remained until released from the Golgi in post-TGN carriers (Fig. 3, D–N). Thus, three stages of traffic in the Golgi area can be distinguished, throughout which PC-I and VSVG colocalize nearly perfectly (see below) and transit together at the same rates. The degree of overlap of each of the two cargoes with the markers of the three Golgi subcompartments during passage through the Golgi can be quantified. The results (Fig. 3, O–Q) underscore once again the remarkable coupling between the movements of the two cargoes, and confirm with a high degree of precision that VSVG and PC-I move together through the transport pathway. Similar results were obtained, with higher spatial resolution, in immuno-EM experiments (see Figs. 5 and 6 and related text).

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