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A novel role for dp115 in the organization of tER sites in Drosophila.

Kondylis V, Rabouille C - J. Cell Biol. (2003)

Bottom Line: However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays.The effects were specific for dp115.Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Center for Cell Biology, Institute for Cell and Molecular Biology, University of Edinburgh, UK.

ABSTRACT
Here, we describe that depletion of the Drosophila homologue of p115 (dp115) by RNA interference in Drosophila S2 cells led to important morphological changes in the Golgi stack morphology and the transitional ER (tER) organization. Using conventional and immunoelectron microscopy and confocal immunofluorescence microscopy, we show that Golgi stacks were converted into clusters of vesicles and tubules, and that the tERs (marked by Sec23p) lost their focused organization and were now dispersed throughout the cytoplasm. However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays. The effects were specific for dp115. Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p. Taken together, these studies suggest that dp115 could be implicated in the architecture of both the Golgi stacks and the tER sites.

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dp115 in Drosophila S2 cells. (A) dGM130 and dp115 proteins were compared with their rat homologues, and the domains of highest homology are painted in dark gray. (B) Western blotting using the affinity-purified dp115/584 of total S2 cell extract corresponding to 2,000,000 cells (lane 1), 4% of the cytosolic fraction of 10,000,000 cells (lane 2), the total membrane corresponding to 10,000,000 cells (lane 3), total extract corresponding to 20 embryos (lane 4), one third instar larva (lane 5). Two bands were recognized (small arrows on the right). Molecular mass markers are indicated on the left. (C–F) IEM of dp115 on Drosophila cells and tissues. Cryosections of PFA (C and E) and PFA/GA (F) fixed S2 cells and Drosophila third instar larvae salivary glands (D) were incubated with the affinity-purified dp115/584 and 10-nm protein A gold. (E) S2 cell sections were double labeled with the dp115/584 antibody followed by 10-nm protein A gold, and the Sec23p antibody followed by 15-nm protein A gold. (F) The same labeling was performed but the gold sizes are inverted. Golgi stacks are marked by a G, and pleiomorphic membranes are marked by an asterisk in C and D. The arrow indicates dp115 in an ER bud in F. Bars, 200 nm.
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fig1: dp115 in Drosophila S2 cells. (A) dGM130 and dp115 proteins were compared with their rat homologues, and the domains of highest homology are painted in dark gray. (B) Western blotting using the affinity-purified dp115/584 of total S2 cell extract corresponding to 2,000,000 cells (lane 1), 4% of the cytosolic fraction of 10,000,000 cells (lane 2), the total membrane corresponding to 10,000,000 cells (lane 3), total extract corresponding to 20 embryos (lane 4), one third instar larva (lane 5). Two bands were recognized (small arrows on the right). Molecular mass markers are indicated on the left. (C–F) IEM of dp115 on Drosophila cells and tissues. Cryosections of PFA (C and E) and PFA/GA (F) fixed S2 cells and Drosophila third instar larvae salivary glands (D) were incubated with the affinity-purified dp115/584 and 10-nm protein A gold. (E) S2 cell sections were double labeled with the dp115/584 antibody followed by 10-nm protein A gold, and the Sec23p antibody followed by 15-nm protein A gold. (F) The same labeling was performed but the gold sizes are inverted. Golgi stacks are marked by a G, and pleiomorphic membranes are marked by an asterisk in C and D. The arrow indicates dp115 in an ER bud in F. Bars, 200 nm.

Mentions: Drosophila p115 (dp115) exhibits 60% similarity to its rat counterpart (Fig. 1 A). dp115 does not possess the acidic stretch of the 50 COOH-terminal amino acids that has been shown in rat p115 to be involved in binding GM130 and giantin (Dirac-Svejstrup et al., 2000). In dp115, there are only a few acidic amino acids scattered along the last 100 COOH-terminal portion of the protein. Besides Neurospora, the tail of Caenorhabditis elegans and Arabidopsis p115 homologues is not acidic either, except for the last 5 and 15 amino acids, respectively (our search). However, the domain in GM130 that binds p115 is conserved in dGM130 (Kondylis et al., 2001; Fig. 1 A), suggesting that both Drosophila proteins could bind each other (albeit possibly through a different domain in dp115).


A novel role for dp115 in the organization of tER sites in Drosophila.

Kondylis V, Rabouille C - J. Cell Biol. (2003)

dp115 in Drosophila S2 cells. (A) dGM130 and dp115 proteins were compared with their rat homologues, and the domains of highest homology are painted in dark gray. (B) Western blotting using the affinity-purified dp115/584 of total S2 cell extract corresponding to 2,000,000 cells (lane 1), 4% of the cytosolic fraction of 10,000,000 cells (lane 2), the total membrane corresponding to 10,000,000 cells (lane 3), total extract corresponding to 20 embryos (lane 4), one third instar larva (lane 5). Two bands were recognized (small arrows on the right). Molecular mass markers are indicated on the left. (C–F) IEM of dp115 on Drosophila cells and tissues. Cryosections of PFA (C and E) and PFA/GA (F) fixed S2 cells and Drosophila third instar larvae salivary glands (D) were incubated with the affinity-purified dp115/584 and 10-nm protein A gold. (E) S2 cell sections were double labeled with the dp115/584 antibody followed by 10-nm protein A gold, and the Sec23p antibody followed by 15-nm protein A gold. (F) The same labeling was performed but the gold sizes are inverted. Golgi stacks are marked by a G, and pleiomorphic membranes are marked by an asterisk in C and D. The arrow indicates dp115 in an ER bud in F. Bars, 200 nm.
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fig1: dp115 in Drosophila S2 cells. (A) dGM130 and dp115 proteins were compared with their rat homologues, and the domains of highest homology are painted in dark gray. (B) Western blotting using the affinity-purified dp115/584 of total S2 cell extract corresponding to 2,000,000 cells (lane 1), 4% of the cytosolic fraction of 10,000,000 cells (lane 2), the total membrane corresponding to 10,000,000 cells (lane 3), total extract corresponding to 20 embryos (lane 4), one third instar larva (lane 5). Two bands were recognized (small arrows on the right). Molecular mass markers are indicated on the left. (C–F) IEM of dp115 on Drosophila cells and tissues. Cryosections of PFA (C and E) and PFA/GA (F) fixed S2 cells and Drosophila third instar larvae salivary glands (D) were incubated with the affinity-purified dp115/584 and 10-nm protein A gold. (E) S2 cell sections were double labeled with the dp115/584 antibody followed by 10-nm protein A gold, and the Sec23p antibody followed by 15-nm protein A gold. (F) The same labeling was performed but the gold sizes are inverted. Golgi stacks are marked by a G, and pleiomorphic membranes are marked by an asterisk in C and D. The arrow indicates dp115 in an ER bud in F. Bars, 200 nm.
Mentions: Drosophila p115 (dp115) exhibits 60% similarity to its rat counterpart (Fig. 1 A). dp115 does not possess the acidic stretch of the 50 COOH-terminal amino acids that has been shown in rat p115 to be involved in binding GM130 and giantin (Dirac-Svejstrup et al., 2000). In dp115, there are only a few acidic amino acids scattered along the last 100 COOH-terminal portion of the protein. Besides Neurospora, the tail of Caenorhabditis elegans and Arabidopsis p115 homologues is not acidic either, except for the last 5 and 15 amino acids, respectively (our search). However, the domain in GM130 that binds p115 is conserved in dGM130 (Kondylis et al., 2001; Fig. 1 A), suggesting that both Drosophila proteins could bind each other (albeit possibly through a different domain in dp115).

Bottom Line: However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays.The effects were specific for dp115.Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Center for Cell Biology, Institute for Cell and Molecular Biology, University of Edinburgh, UK.

ABSTRACT
Here, we describe that depletion of the Drosophila homologue of p115 (dp115) by RNA interference in Drosophila S2 cells led to important morphological changes in the Golgi stack morphology and the transitional ER (tER) organization. Using conventional and immunoelectron microscopy and confocal immunofluorescence microscopy, we show that Golgi stacks were converted into clusters of vesicles and tubules, and that the tERs (marked by Sec23p) lost their focused organization and were now dispersed throughout the cytoplasm. However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays. The effects were specific for dp115. Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p. Taken together, these studies suggest that dp115 could be implicated in the architecture of both the Golgi stacks and the tER sites.

Show MeSH
Related in: MedlinePlus