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Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature.

Ling Y, Hayano S, Novick P - Mol. Biol. Cell (2014)

Bottom Line: Phosphatidylinositol-4-phosphate (PI4P) is produced on both the Golgi and the plasma membrane.We show here that in yeast the oxysterol-binding proteins Osh1-Osh7 are collectively needed to maintain the normal distribution of PI4P and that Osh4p is critical in this function.This reduction in PI4P is necessary for a switch in the regulation of the Sec4p exchange protein, Sec2p, from an interaction with the upstream Rab, Ypt31/32, to an interaction with a downstream Sec4p effector, Sec15p.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093.

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Osh4 positively regulates the Sec2–Sec15 interaction. (A) The Sec2–Sec15 interaction is inhibited in osh4Δ cells and sac1Δ cells. Sec2-3xGFP was immunoprecipitated with anti-GFP antibody from wild-type, osh4Δ, or sac1Δ cell lysates. As a negative control, wild-type cells expressing nontagged Sec2 were included in the coimmunoprecipitation experiments. We loaded 0.5% of whole-cell lysates as input and processed the rest of the samples for coimmunoprecipitation. Sec2-3xGFP in the immunoprecipitates was detected with anti-Sec2 antibody. Coprecipitated Sec15-13xmyc was detected with anti-myc antibody. (B) Quantification of the Sec2–Sec15 interaction in wild-type, osh4Δ, and sac1Δ cells. Three independent experiments were performed. The intensity of the bands was quantified using ImageJ. The percentage of Sec15 in the immunoprecipitate was calculated and is indicated. Mean and SD of three experiments.
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Figure 6: Osh4 positively regulates the Sec2–Sec15 interaction. (A) The Sec2–Sec15 interaction is inhibited in osh4Δ cells and sac1Δ cells. Sec2-3xGFP was immunoprecipitated with anti-GFP antibody from wild-type, osh4Δ, or sac1Δ cell lysates. As a negative control, wild-type cells expressing nontagged Sec2 were included in the coimmunoprecipitation experiments. We loaded 0.5% of whole-cell lysates as input and processed the rest of the samples for coimmunoprecipitation. Sec2-3xGFP in the immunoprecipitates was detected with anti-Sec2 antibody. Coprecipitated Sec15-13xmyc was detected with anti-myc antibody. (B) Quantification of the Sec2–Sec15 interaction in wild-type, osh4Δ, and sac1Δ cells. Three independent experiments were performed. The intensity of the bands was quantified using ImageJ. The percentage of Sec15 in the immunoprecipitate was calculated and is indicated. Mean and SD of three experiments.

Mentions: PI4P inhibits the interaction between Sec2p and Sec15p both in vivo and in vitro (Mizuno-Yamasaki et al., 2010); therefore we investigated whether this interaction is also controlled by Osh4p in vivo. We tagged Sec2p with 3xGFP and Sec15p with 13myc in wild-type, osh4Δ, and sac1Δ cells. We performed coimmunoprecipitation experiments, pulling down Sec2-3xGFP with the anti-GFP antibody and then probing for Sec15p with anti-myc antibody. Consistent with the microscopy data, we observed that in both osh4Δ and sac1Δ cells, less Sec15-13myc was coimmunoprecipitated than with wild-type cells (Figure 6A). Quantification of the coprecipitation experiment revealed that the interaction between Sec2p and Sec15p was roughly threefold lower on average in osh4Δ and sac1Δ cells than with wild-type cells (Figure 6B). These data further support the model that Osh4p negatively regulates PI4P levels of secretory vesicles and high levels of PI4P in osh4 Δ cells inhibits the interaction between Sec2p and Sec15p.


Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature.

Ling Y, Hayano S, Novick P - Mol. Biol. Cell (2014)

Osh4 positively regulates the Sec2–Sec15 interaction. (A) The Sec2–Sec15 interaction is inhibited in osh4Δ cells and sac1Δ cells. Sec2-3xGFP was immunoprecipitated with anti-GFP antibody from wild-type, osh4Δ, or sac1Δ cell lysates. As a negative control, wild-type cells expressing nontagged Sec2 were included in the coimmunoprecipitation experiments. We loaded 0.5% of whole-cell lysates as input and processed the rest of the samples for coimmunoprecipitation. Sec2-3xGFP in the immunoprecipitates was detected with anti-Sec2 antibody. Coprecipitated Sec15-13xmyc was detected with anti-myc antibody. (B) Quantification of the Sec2–Sec15 interaction in wild-type, osh4Δ, and sac1Δ cells. Three independent experiments were performed. The intensity of the bands was quantified using ImageJ. The percentage of Sec15 in the immunoprecipitate was calculated and is indicated. Mean and SD of three experiments.
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Related In: Results  -  Collection

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Figure 6: Osh4 positively regulates the Sec2–Sec15 interaction. (A) The Sec2–Sec15 interaction is inhibited in osh4Δ cells and sac1Δ cells. Sec2-3xGFP was immunoprecipitated with anti-GFP antibody from wild-type, osh4Δ, or sac1Δ cell lysates. As a negative control, wild-type cells expressing nontagged Sec2 were included in the coimmunoprecipitation experiments. We loaded 0.5% of whole-cell lysates as input and processed the rest of the samples for coimmunoprecipitation. Sec2-3xGFP in the immunoprecipitates was detected with anti-Sec2 antibody. Coprecipitated Sec15-13xmyc was detected with anti-myc antibody. (B) Quantification of the Sec2–Sec15 interaction in wild-type, osh4Δ, and sac1Δ cells. Three independent experiments were performed. The intensity of the bands was quantified using ImageJ. The percentage of Sec15 in the immunoprecipitate was calculated and is indicated. Mean and SD of three experiments.
Mentions: PI4P inhibits the interaction between Sec2p and Sec15p both in vivo and in vitro (Mizuno-Yamasaki et al., 2010); therefore we investigated whether this interaction is also controlled by Osh4p in vivo. We tagged Sec2p with 3xGFP and Sec15p with 13myc in wild-type, osh4Δ, and sac1Δ cells. We performed coimmunoprecipitation experiments, pulling down Sec2-3xGFP with the anti-GFP antibody and then probing for Sec15p with anti-myc antibody. Consistent with the microscopy data, we observed that in both osh4Δ and sac1Δ cells, less Sec15-13myc was coimmunoprecipitated than with wild-type cells (Figure 6A). Quantification of the coprecipitation experiment revealed that the interaction between Sec2p and Sec15p was roughly threefold lower on average in osh4Δ and sac1Δ cells than with wild-type cells (Figure 6B). These data further support the model that Osh4p negatively regulates PI4P levels of secretory vesicles and high levels of PI4P in osh4 Δ cells inhibits the interaction between Sec2p and Sec15p.

Bottom Line: Phosphatidylinositol-4-phosphate (PI4P) is produced on both the Golgi and the plasma membrane.We show here that in yeast the oxysterol-binding proteins Osh1-Osh7 are collectively needed to maintain the normal distribution of PI4P and that Osh4p is critical in this function.This reduction in PI4P is necessary for a switch in the regulation of the Sec4p exchange protein, Sec2p, from an interaction with the upstream Rab, Ypt31/32, to an interaction with a downstream Sec4p effector, Sec15p.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093.

Show MeSH
Related in: MedlinePlus