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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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Depletion of dGM130 protein and dp115 mRNA. (A) Western blotting using MLO7 (anti-GM130 antibody) of the extract of S2 cells incubated with (+) or without (−) ds dGM130 for increasing lengths of time. C corresponds to time 0 (3,000,000 cells). 1,500,000 cells were used for lanes 24–72 h, and 2,500,000 for lanes 96–120 h. From the two bands the antibody recognizes, the stronger upper band is specifically depleted (arrow). (B) The dp115 mRNA was measured by RT-PCR from total RNA extract from 1,000,000 cells incubated with (+) or without (−) ds dp115 for 48–120 h. Amplification of histone 2A mRNA was used as control of the specific depletion of dp115 mRNA and as a loading control. Western blotting of the extract of cells (1,500,000) incubated with (+) or without (−) ds dp115 for 72 and 96 h using the dp115/584 antibody (dp115), the Sec23p antibody (dSec23p), the antibody recognizing the 120-kD Drosophila antigen (d120kd), and α-tubulin. Note that only dp115 is depleted.
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fig2: Depletion of dGM130 protein and dp115 mRNA. (A) Western blotting using MLO7 (anti-GM130 antibody) of the extract of S2 cells incubated with (+) or without (−) ds dGM130 for increasing lengths of time. C corresponds to time 0 (3,000,000 cells). 1,500,000 cells were used for lanes 24–72 h, and 2,500,000 for lanes 96–120 h. From the two bands the antibody recognizes, the stronger upper band is specifically depleted (arrow). (B) The dp115 mRNA was measured by RT-PCR from total RNA extract from 1,000,000 cells incubated with (+) or without (−) ds dp115 for 48–120 h. Amplification of histone 2A mRNA was used as control of the specific depletion of dp115 mRNA and as a loading control. Western blotting of the extract of cells (1,500,000) incubated with (+) or without (−) ds dp115 for 72 and 96 h using the dp115/584 antibody (dp115), the Sec23p antibody (dSec23p), the antibody recognizing the 120-kD Drosophila antigen (d120kd), and α-tubulin. Note that only dp115 is depleted.

Mentions: We depleted S2 cells of dGM130 using a dsRNA corresponding to the second exon of dGM130 (ds dGM130). When samples were analyzed by Western blotting, two bands were detected, one very faint, which was neglected (Fig. 2, top, lane C), and a strong one. The strong band migrated at the predicted position for a protein of the mass of dGM130 (arrow) and was reduced below detectable level after 48 h (Fig. 2, top) and remained so up to 120 h of incubation. This depletion, however, did not lead to any effect on Golgi stack morphology, as assessed by EM (Fig. 3 B).


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

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

Depletion of dGM130 protein and dp115 mRNA. (A) Western blotting using MLO7 (anti-GM130 antibody) of the extract of S2 cells incubated with (+) or without (−) ds dGM130 for increasing lengths of time. C corresponds to time 0 (3,000,000 cells). 1,500,000 cells were used for lanes 24–72 h, and 2,500,000 for lanes 96–120 h. From the two bands the antibody recognizes, the stronger upper band is specifically depleted (arrow). (B) The dp115 mRNA was measured by RT-PCR from total RNA extract from 1,000,000 cells incubated with (+) or without (−) ds dp115 for 48–120 h. Amplification of histone 2A mRNA was used as control of the specific depletion of dp115 mRNA and as a loading control. Western blotting of the extract of cells (1,500,000) incubated with (+) or without (−) ds dp115 for 72 and 96 h using the dp115/584 antibody (dp115), the Sec23p antibody (dSec23p), the antibody recognizing the 120-kD Drosophila antigen (d120kd), and α-tubulin. Note that only dp115 is depleted.
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Related In: Results  -  Collection

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fig2: Depletion of dGM130 protein and dp115 mRNA. (A) Western blotting using MLO7 (anti-GM130 antibody) of the extract of S2 cells incubated with (+) or without (−) ds dGM130 for increasing lengths of time. C corresponds to time 0 (3,000,000 cells). 1,500,000 cells were used for lanes 24–72 h, and 2,500,000 for lanes 96–120 h. From the two bands the antibody recognizes, the stronger upper band is specifically depleted (arrow). (B) The dp115 mRNA was measured by RT-PCR from total RNA extract from 1,000,000 cells incubated with (+) or without (−) ds dp115 for 48–120 h. Amplification of histone 2A mRNA was used as control of the specific depletion of dp115 mRNA and as a loading control. Western blotting of the extract of cells (1,500,000) incubated with (+) or without (−) ds dp115 for 72 and 96 h using the dp115/584 antibody (dp115), the Sec23p antibody (dSec23p), the antibody recognizing the 120-kD Drosophila antigen (d120kd), and α-tubulin. Note that only dp115 is depleted.
Mentions: We depleted S2 cells of dGM130 using a dsRNA corresponding to the second exon of dGM130 (ds dGM130). When samples were analyzed by Western blotting, two bands were detected, one very faint, which was neglected (Fig. 2, top, lane C), and a strong one. The strong band migrated at the predicted position for a protein of the mass of dGM130 (arrow) and was reduced below detectable level after 48 h (Fig. 2, top) and remained so up to 120 h of incubation. This depletion, however, did not lead to any effect on Golgi stack morphology, as assessed by EM (Fig. 3 B).

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