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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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Related in: MedlinePlus

PC-I and VSVG move through the main Golgi subcompartments (cis-, medial-, and trans-TGN) at indistinguishable rates. Human fibroblasts were fixed at steady state (A–C) or subjected to the small-pulse protocol and fixed at various times after release of the 15°C block (D–N). (A–C) Golgi areas stained for GM130 (A, red) and TGN (B, green); the merged image is shown in C. Note that the patterns of two colors appear very similar in (A) and (B), but they clearly do not overlap (C). (D–N) Cells subjected to the small-pulse protocol were fixed at the times indicated in the figure after releasing the 15°C block and double labeled for VSVG and GM130 (D, G, and L), VSVG and GT (e, h, and m), or VSVG and TGN (f, i, and n). VSVG is red and GM130, GT, and TGN are green. At time 0, VSVG localized in peripheral spots (IC elements) and did not overlap with the Golgi markers (unpublished data). At 3.5 min, VSVG colocalized with GM130 (D) but not with GT (E) and TGN46 (F). Later (7 min), VSVG lost colocalization with GM130 (G) acquired colocalization with GT (H), and did not colocalize with TGN46 (I). Finally (11 min), VSVG lost colocalization with GM130 (L) and GT (M) and acquired colocalization with TGN46 (N). Identical results were obtained by labeling PC-I instead of VSVG (see below), and the two cargoes colocalized perfectly (unpublished data). (O–Q) Quantification and time course of the passage of VSVG and PC-I through the main Golgi subcompartments. The localization of cargoes in each subcompartment was assessed by measuring the degree of overlap of each cargo with the marker of each subcompartment (GM130, GT, and TGN) (see Materials and methods), and is expressed as the percentage of colocalization (percentage of cargo-containing pixels which also contain the appropriate Golgi marker). It is apparent that the two cargoes move together. Each value represents the average of eight to fourteen independent measurements from at least three different experiments. The SDs did not exceed 15% of the mean. Bar: (A–C) 12 μm; (D, F–I, L, and M) 10 μm; (E and H) μm 7,5; (N) 5 μm.
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fig3: PC-I and VSVG move through the main Golgi subcompartments (cis-, medial-, and trans-TGN) at indistinguishable rates. Human fibroblasts were fixed at steady state (A–C) or subjected to the small-pulse protocol and fixed at various times after release of the 15°C block (D–N). (A–C) Golgi areas stained for GM130 (A, red) and TGN (B, green); the merged image is shown in C. Note that the patterns of two colors appear very similar in (A) and (B), but they clearly do not overlap (C). (D–N) Cells subjected to the small-pulse protocol were fixed at the times indicated in the figure after releasing the 15°C block and double labeled for VSVG and GM130 (D, G, and L), VSVG and GT (e, h, and m), or VSVG and TGN (f, i, and n). VSVG is red and GM130, GT, and TGN are green. At time 0, VSVG localized in peripheral spots (IC elements) and did not overlap with the Golgi markers (unpublished data). At 3.5 min, VSVG colocalized with GM130 (D) but not with GT (E) and TGN46 (F). Later (7 min), VSVG lost colocalization with GM130 (G) acquired colocalization with GT (H), and did not colocalize with TGN46 (I). Finally (11 min), VSVG lost colocalization with GM130 (L) and GT (M) and acquired colocalization with TGN46 (N). Identical results were obtained by labeling PC-I instead of VSVG (see below), and the two cargoes colocalized perfectly (unpublished data). (O–Q) Quantification and time course of the passage of VSVG and PC-I through the main Golgi subcompartments. The localization of cargoes in each subcompartment was assessed by measuring the degree of overlap of each cargo with the marker of each subcompartment (GM130, GT, and TGN) (see Materials and methods), and is expressed as the percentage of colocalization (percentage of cargo-containing pixels which also contain the appropriate Golgi marker). It is apparent that the two cargoes move together. Each value represents the average of eight to fourteen independent measurements from at least three different experiments. The SDs did not exceed 15% of the mean. Bar: (A–C) 12 μm; (D, F–I, L, and M) 10 μm; (E and H) μm 7,5; (N) 5 μm.

Mentions: The amount of cargo reaching the Golgi per unit time in this experiment is probably much larger than during physiological traffic. To examine the behavior of the transport pathway under conditions closer to normal, we used the small-pulse protocol in which the accumulation of cargo in the IC is reduced but still sufficient to allow the formation of visible cargo-containing IC elements (scheme in Fig. 1). In spite of some fluorescence background due to cargo remaining in the ER, it was clear that, after release of the 15°C block, PC-I and VSVG colocalized and reached the Golgi simultaneously within 3–4 min, resided in the Golgi area for ∼8 min (transit time, legend to Fig. 2), and then left it, again simultaneously (Fig. 2, L–N). Thus, the movements of PC-I and VSVG could not be dissociated, even using this protocol. Notably, the cargoes in the Golgi area were discontinuously distributed in 10–30 discrete fluorescent spots of variable intensities (Fig. 3 and see Fig. 8). Also, interestingly, the transit time of the two cargoes through the Golgi area was shorter than when using the large-pulse protocol (8 vs. 20 min; quantification in Fig. 2, O and Q), suggesting that the transport behavior of the Golgi is influenced by the amount of cargo processed per unit time. This cargo effect is being analyzed separately.


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 move through the main Golgi subcompartments (cis-, medial-, and trans-TGN) at indistinguishable rates. Human fibroblasts were fixed at steady state (A–C) or subjected to the small-pulse protocol and fixed at various times after release of the 15°C block (D–N). (A–C) Golgi areas stained for GM130 (A, red) and TGN (B, green); the merged image is shown in C. Note that the patterns of two colors appear very similar in (A) and (B), but they clearly do not overlap (C). (D–N) Cells subjected to the small-pulse protocol were fixed at the times indicated in the figure after releasing the 15°C block and double labeled for VSVG and GM130 (D, G, and L), VSVG and GT (e, h, and m), or VSVG and TGN (f, i, and n). VSVG is red and GM130, GT, and TGN are green. At time 0, VSVG localized in peripheral spots (IC elements) and did not overlap with the Golgi markers (unpublished data). At 3.5 min, VSVG colocalized with GM130 (D) but not with GT (E) and TGN46 (F). Later (7 min), VSVG lost colocalization with GM130 (G) acquired colocalization with GT (H), and did not colocalize with TGN46 (I). Finally (11 min), VSVG lost colocalization with GM130 (L) and GT (M) and acquired colocalization with TGN46 (N). Identical results were obtained by labeling PC-I instead of VSVG (see below), and the two cargoes colocalized perfectly (unpublished data). (O–Q) Quantification and time course of the passage of VSVG and PC-I through the main Golgi subcompartments. The localization of cargoes in each subcompartment was assessed by measuring the degree of overlap of each cargo with the marker of each subcompartment (GM130, GT, and TGN) (see Materials and methods), and is expressed as the percentage of colocalization (percentage of cargo-containing pixels which also contain the appropriate Golgi marker). It is apparent that the two cargoes move together. Each value represents the average of eight to fourteen independent measurements from at least three different experiments. The SDs did not exceed 15% of the mean. Bar: (A–C) 12 μm; (D, F–I, L, and M) 10 μm; (E and H) μm 7,5; (N) 5 μm.
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fig3: PC-I and VSVG move through the main Golgi subcompartments (cis-, medial-, and trans-TGN) at indistinguishable rates. Human fibroblasts were fixed at steady state (A–C) or subjected to the small-pulse protocol and fixed at various times after release of the 15°C block (D–N). (A–C) Golgi areas stained for GM130 (A, red) and TGN (B, green); the merged image is shown in C. Note that the patterns of two colors appear very similar in (A) and (B), but they clearly do not overlap (C). (D–N) Cells subjected to the small-pulse protocol were fixed at the times indicated in the figure after releasing the 15°C block and double labeled for VSVG and GM130 (D, G, and L), VSVG and GT (e, h, and m), or VSVG and TGN (f, i, and n). VSVG is red and GM130, GT, and TGN are green. At time 0, VSVG localized in peripheral spots (IC elements) and did not overlap with the Golgi markers (unpublished data). At 3.5 min, VSVG colocalized with GM130 (D) but not with GT (E) and TGN46 (F). Later (7 min), VSVG lost colocalization with GM130 (G) acquired colocalization with GT (H), and did not colocalize with TGN46 (I). Finally (11 min), VSVG lost colocalization with GM130 (L) and GT (M) and acquired colocalization with TGN46 (N). Identical results were obtained by labeling PC-I instead of VSVG (see below), and the two cargoes colocalized perfectly (unpublished data). (O–Q) Quantification and time course of the passage of VSVG and PC-I through the main Golgi subcompartments. The localization of cargoes in each subcompartment was assessed by measuring the degree of overlap of each cargo with the marker of each subcompartment (GM130, GT, and TGN) (see Materials and methods), and is expressed as the percentage of colocalization (percentage of cargo-containing pixels which also contain the appropriate Golgi marker). It is apparent that the two cargoes move together. Each value represents the average of eight to fourteen independent measurements from at least three different experiments. The SDs did not exceed 15% of the mean. Bar: (A–C) 12 μm; (D, F–I, L, and M) 10 μm; (E and H) μm 7,5; (N) 5 μm.
Mentions: The amount of cargo reaching the Golgi per unit time in this experiment is probably much larger than during physiological traffic. To examine the behavior of the transport pathway under conditions closer to normal, we used the small-pulse protocol in which the accumulation of cargo in the IC is reduced but still sufficient to allow the formation of visible cargo-containing IC elements (scheme in Fig. 1). In spite of some fluorescence background due to cargo remaining in the ER, it was clear that, after release of the 15°C block, PC-I and VSVG colocalized and reached the Golgi simultaneously within 3–4 min, resided in the Golgi area for ∼8 min (transit time, legend to Fig. 2), and then left it, again simultaneously (Fig. 2, L–N). Thus, the movements of PC-I and VSVG could not be dissociated, even using this protocol. Notably, the cargoes in the Golgi area were discontinuously distributed in 10–30 discrete fluorescent spots of variable intensities (Fig. 3 and see Fig. 8). Also, interestingly, the transit time of the two cargoes through the Golgi area was shorter than when using the large-pulse protocol (8 vs. 20 min; quantification in Fig. 2, O and Q), suggesting that the transport behavior of the Golgi is influenced by the amount of cargo processed per unit time. This cargo effect is being analyzed separately.

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