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Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

Lehman K, Rossi G, Adamo JE, Brennwald P - J. Cell Biol. (1999)

Bottom Line: In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion.Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase.Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Weill Medical College of Cornell University, New York, New York 10021, USA.

ABSTRACT
We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

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Sro7p interacts specifically with Sec9p in the cytoplasm and at the plasma membrane. (A) Cells containing high copy SEC9 (pB35) were grown to logarithmic phase, spheroplasted, and lysed. The lysate was centrifuged at 30,000 g to separate cytosol and membrane fractions, the samples were adjusted to 0.5% NP-40, centrifuged at 13,000 g for 10 min to eliminate insoluble material, and subjected to coimmunoprecipitation analysis as in Fig. 8. Samples were analyzed by SDS-PAGE and immunoblotted with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p antibodies. (B) Serial coprecipitation analysis demonstrates that Sro7p interacts with Sso1/2p and Snc1/2p only through the interaction with Sec9p. A 30,000 g membrane fraction was solubilized with detergent as above and was precleared of Sec9 protein by immunoprecipitation with saturating amounts of affinity-purified α-Sec9p antibody or equivalent amounts of preimmune IgG as a control. Fractions were subjected to a second round of immunoprecipitations with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p polyclonal antibodies and analyzed as above.
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Figure 10: Sro7p interacts specifically with Sec9p in the cytoplasm and at the plasma membrane. (A) Cells containing high copy SEC9 (pB35) were grown to logarithmic phase, spheroplasted, and lysed. The lysate was centrifuged at 30,000 g to separate cytosol and membrane fractions, the samples were adjusted to 0.5% NP-40, centrifuged at 13,000 g for 10 min to eliminate insoluble material, and subjected to coimmunoprecipitation analysis as in Fig. 8. Samples were analyzed by SDS-PAGE and immunoblotted with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p antibodies. (B) Serial coprecipitation analysis demonstrates that Sro7p interacts with Sso1/2p and Snc1/2p only through the interaction with Sec9p. A 30,000 g membrane fraction was solubilized with detergent as above and was precleared of Sec9 protein by immunoprecipitation with saturating amounts of affinity-purified α-Sec9p antibody or equivalent amounts of preimmune IgG as a control. Fractions were subjected to a second round of immunoprecipitations with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p polyclonal antibodies and analyzed as above.

Mentions: Since both Sec9p and Sro7p have significant pools of protein on both the plasma membrane and in the cytosol, we examined whether the interaction seen between the two proteins occurred in one or both pools. For these experiments, it was necessary to use yeast cells expressing Sec9p on a multicopy plasmid (which results in about a fivefold increase in Sec9p levels) to readily detect the interaction after the separation of the cytosol and membrane fractions. Lysates were centrifuged at 30,000 g to separate the soluble and membrane fractions, and then each was diluted 1:1 with lysis buffer containing 1% NP-40 to solubilize the membranes. The samples were subjected to immunoprecipitation exactly as for the whole cell lysates. The results, shown in Fig. 10 A, show that Sro7p and Sec9p can coprecipitate each other in both the membrane and supernatant fractions of the cell. As expected the transmembrane SNARE proteins Sso1/2 and Snc1/2 are predominantly found in association with each other in the 30,000 g membrane fraction. However, a significant amount of association between Sro7p and Sec9p was seen in both the supernatant and membrane fractions.


Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

Lehman K, Rossi G, Adamo JE, Brennwald P - J. Cell Biol. (1999)

Sro7p interacts specifically with Sec9p in the cytoplasm and at the plasma membrane. (A) Cells containing high copy SEC9 (pB35) were grown to logarithmic phase, spheroplasted, and lysed. The lysate was centrifuged at 30,000 g to separate cytosol and membrane fractions, the samples were adjusted to 0.5% NP-40, centrifuged at 13,000 g for 10 min to eliminate insoluble material, and subjected to coimmunoprecipitation analysis as in Fig. 8. Samples were analyzed by SDS-PAGE and immunoblotted with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p antibodies. (B) Serial coprecipitation analysis demonstrates that Sro7p interacts with Sso1/2p and Snc1/2p only through the interaction with Sec9p. A 30,000 g membrane fraction was solubilized with detergent as above and was precleared of Sec9 protein by immunoprecipitation with saturating amounts of affinity-purified α-Sec9p antibody or equivalent amounts of preimmune IgG as a control. Fractions were subjected to a second round of immunoprecipitations with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p polyclonal antibodies and analyzed as above.
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Related In: Results  -  Collection

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Figure 10: Sro7p interacts specifically with Sec9p in the cytoplasm and at the plasma membrane. (A) Cells containing high copy SEC9 (pB35) were grown to logarithmic phase, spheroplasted, and lysed. The lysate was centrifuged at 30,000 g to separate cytosol and membrane fractions, the samples were adjusted to 0.5% NP-40, centrifuged at 13,000 g for 10 min to eliminate insoluble material, and subjected to coimmunoprecipitation analysis as in Fig. 8. Samples were analyzed by SDS-PAGE and immunoblotted with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p antibodies. (B) Serial coprecipitation analysis demonstrates that Sro7p interacts with Sso1/2p and Snc1/2p only through the interaction with Sec9p. A 30,000 g membrane fraction was solubilized with detergent as above and was precleared of Sec9 protein by immunoprecipitation with saturating amounts of affinity-purified α-Sec9p antibody or equivalent amounts of preimmune IgG as a control. Fractions were subjected to a second round of immunoprecipitations with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p polyclonal antibodies and analyzed as above.
Mentions: Since both Sec9p and Sro7p have significant pools of protein on both the plasma membrane and in the cytosol, we examined whether the interaction seen between the two proteins occurred in one or both pools. For these experiments, it was necessary to use yeast cells expressing Sec9p on a multicopy plasmid (which results in about a fivefold increase in Sec9p levels) to readily detect the interaction after the separation of the cytosol and membrane fractions. Lysates were centrifuged at 30,000 g to separate the soluble and membrane fractions, and then each was diluted 1:1 with lysis buffer containing 1% NP-40 to solubilize the membranes. The samples were subjected to immunoprecipitation exactly as for the whole cell lysates. The results, shown in Fig. 10 A, show that Sro7p and Sec9p can coprecipitate each other in both the membrane and supernatant fractions of the cell. As expected the transmembrane SNARE proteins Sso1/2 and Snc1/2 are predominantly found in association with each other in the 30,000 g membrane fraction. However, a significant amount of association between Sro7p and Sec9p was seen in both the supernatant and membrane fractions.

Bottom Line: In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion.Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase.Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Weill Medical College of Cornell University, New York, New York 10021, USA.

ABSTRACT
We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

Show MeSH
Related in: MedlinePlus