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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 is found in both cytosolic- and membrane-bound pools. (A) Affinity-purified antibodies to Sro7p recognize a protein of ∼105 kD on SDS-PAGE. Cells from wild-type (SRO7, SRO77), single disruptants (sro77Δ or sro7Δ), double disruptants (sro77Δ, sro7Δ), and strains containing high copy SRO7 (2μ SRO7) were spheroplasted, lysed, and boiled in SDS sample buffer. The samples were examined by 7% SDS-PAGE gels followed by immunoblotting with affinity-purified α-Sro7p antibodies. The higher molecular mass forms present in the SRO7 high copy lane probably represent denatured aggregates since they are much less apparent when samples are diluted before boiling and do not change appreciably during pulse-chase experiments (data not shown). (B) Sro7p is found in the soluble and membrane fractions of the cell. Cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown to logarithmic phase, spheroplasted, lysed, and spun to remove unbroken cells. The lysate was treated with detergent or a mock control and subjected to two successive centrifugations: 30,000 g for 15 min, and then the S30 supernatant was centrifuged at 100,000 g for 1 h. Pellet fractions were resuspended in the same volumes as the supernatants to normalize. Samples from each fraction were boiled, analyzed by 7% SDS-PAGE, and immunoblotted with affinity-purified α-Sro7p antibody. Samples were also run on a 12.5% gel and immunoblotted with α-Sso1/2p polyclonal antibody as an internal control of the fractionation procedure.
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Figure 5: Sro7p is found in both cytosolic- and membrane-bound pools. (A) Affinity-purified antibodies to Sro7p recognize a protein of ∼105 kD on SDS-PAGE. Cells from wild-type (SRO7, SRO77), single disruptants (sro77Δ or sro7Δ), double disruptants (sro77Δ, sro7Δ), and strains containing high copy SRO7 (2μ SRO7) were spheroplasted, lysed, and boiled in SDS sample buffer. The samples were examined by 7% SDS-PAGE gels followed by immunoblotting with affinity-purified α-Sro7p antibodies. The higher molecular mass forms present in the SRO7 high copy lane probably represent denatured aggregates since they are much less apparent when samples are diluted before boiling and do not change appreciably during pulse-chase experiments (data not shown). (B) Sro7p is found in the soluble and membrane fractions of the cell. Cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown to logarithmic phase, spheroplasted, lysed, and spun to remove unbroken cells. The lysate was treated with detergent or a mock control and subjected to two successive centrifugations: 30,000 g for 15 min, and then the S30 supernatant was centrifuged at 100,000 g for 1 h. Pellet fractions were resuspended in the same volumes as the supernatants to normalize. Samples from each fraction were boiled, analyzed by 7% SDS-PAGE, and immunoblotted with affinity-purified α-Sro7p antibody. Samples were also run on a 12.5% gel and immunoblotted with α-Sso1/2p polyclonal antibody as an internal control of the fractionation procedure.

Mentions: Since Sro7p/77p both bind to Sec9p and function at the same stage of exocytosis, we reasoned that the localization of these proteins within the cell might overlap with that of Sec9p. To examine the Sro7 protein function within yeast cells and determine its subcellular localization, we raised antibodies to the COOH-terminal 219 residues of the protein (see Materials and Methods). The affinity-purified antibodies recognize a protein of ∼105 kD that corresponds to the Sro7 protein, as it is absent in a strain that lacks Sro7 and is greatly enhanced in cells carrying a plasmid overexpressing the Sro7 protein (Fig. 5 A).


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 is found in both cytosolic- and membrane-bound pools. (A) Affinity-purified antibodies to Sro7p recognize a protein of ∼105 kD on SDS-PAGE. Cells from wild-type (SRO7, SRO77), single disruptants (sro77Δ or sro7Δ), double disruptants (sro77Δ, sro7Δ), and strains containing high copy SRO7 (2μ SRO7) were spheroplasted, lysed, and boiled in SDS sample buffer. The samples were examined by 7% SDS-PAGE gels followed by immunoblotting with affinity-purified α-Sro7p antibodies. The higher molecular mass forms present in the SRO7 high copy lane probably represent denatured aggregates since they are much less apparent when samples are diluted before boiling and do not change appreciably during pulse-chase experiments (data not shown). (B) Sro7p is found in the soluble and membrane fractions of the cell. Cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown to logarithmic phase, spheroplasted, lysed, and spun to remove unbroken cells. The lysate was treated with detergent or a mock control and subjected to two successive centrifugations: 30,000 g for 15 min, and then the S30 supernatant was centrifuged at 100,000 g for 1 h. Pellet fractions were resuspended in the same volumes as the supernatants to normalize. Samples from each fraction were boiled, analyzed by 7% SDS-PAGE, and immunoblotted with affinity-purified α-Sro7p antibody. Samples were also run on a 12.5% gel and immunoblotted with α-Sso1/2p polyclonal antibody as an internal control of the fractionation procedure.
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Figure 5: Sro7p is found in both cytosolic- and membrane-bound pools. (A) Affinity-purified antibodies to Sro7p recognize a protein of ∼105 kD on SDS-PAGE. Cells from wild-type (SRO7, SRO77), single disruptants (sro77Δ or sro7Δ), double disruptants (sro77Δ, sro7Δ), and strains containing high copy SRO7 (2μ SRO7) were spheroplasted, lysed, and boiled in SDS sample buffer. The samples were examined by 7% SDS-PAGE gels followed by immunoblotting with affinity-purified α-Sro7p antibodies. The higher molecular mass forms present in the SRO7 high copy lane probably represent denatured aggregates since they are much less apparent when samples are diluted before boiling and do not change appreciably during pulse-chase experiments (data not shown). (B) Sro7p is found in the soluble and membrane fractions of the cell. Cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown to logarithmic phase, spheroplasted, lysed, and spun to remove unbroken cells. The lysate was treated with detergent or a mock control and subjected to two successive centrifugations: 30,000 g for 15 min, and then the S30 supernatant was centrifuged at 100,000 g for 1 h. Pellet fractions were resuspended in the same volumes as the supernatants to normalize. Samples from each fraction were boiled, analyzed by 7% SDS-PAGE, and immunoblotted with affinity-purified α-Sro7p antibody. Samples were also run on a 12.5% gel and immunoblotted with α-Sso1/2p polyclonal antibody as an internal control of the fractionation procedure.
Mentions: Since Sro7p/77p both bind to Sec9p and function at the same stage of exocytosis, we reasoned that the localization of these proteins within the cell might overlap with that of Sec9p. To examine the Sro7 protein function within yeast cells and determine its subcellular localization, we raised antibodies to the COOH-terminal 219 residues of the protein (see Materials and Methods). The affinity-purified antibodies recognize a protein of ∼105 kD that corresponds to the Sro7 protein, as it is absent in a strain that lacks Sro7 and is greatly enhanced in cells carrying a plasmid overexpressing the Sro7 protein (Fig. 5 A).

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