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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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Full-length Sro7p, but not the COOH-terminal half of Sro7p, can be directly cross-linked to Sec9p in yeast lysates. Coprecipitation analysis was performed on radiolabeled yeast strains transformed with either myc-tagged YEpSEC9-myc plasmid (pB37) or, as a control, an otherwise identical untagged YEpSEC9 plasmid (pB37) in strains which contained a construct overexpressing either full-length Sro7 (pB363) or CT Sro7 (pB367) under control of the inducible GAL1 promoter. All strains were induced in galactose-containing media for 2 h before 1 h labeling of cells with 35S-Express label in galactose-containing media. The cells were spheroplasted and lysed osmotically in PBS. Lysates were immediately subjected to treatment with the protein cleavable cross-linking agent (DSP) dissolved in DMSO or a DMSO control for 20 min on ice and subsequently boiled in 1% SDS buffer before dilution with IP buffer. Association because of cross-linking was monitored by a two-step immunoprecipitation protocol with the first immunoprecipitation being with the α-myc or directly with α-Sro7p antibodies (α-Sro7 IP). After washing, samples were boiled in 1% SDS/0.1 M DTT (to cleave the cross-linker), diluted with IP buffer, and subjected to a second round of immunoprecipitation with either α-Sro7p (top) or α-Sec9p (bottom) polyclonal antibodies. Samples were boiled in sample buffer, resolved by SDS-PAGE, dried, and exposed to film. The expression from the GAL1-SRO7 and GAL1-SRO7-CT constructs were similar (compare first and last lanes, marked α-Sro7 IP). A cross-linker and myc tag–dependent interaction is apparent between full-length Sro7p and Sec9p, strongly suggesting that these proteins are directly associated with each other in vivo. In contrast, the COOH-terminal domain does not show a detectable cross-linking in this assay.
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Figure 8: Full-length Sro7p, but not the COOH-terminal half of Sro7p, can be directly cross-linked to Sec9p in yeast lysates. Coprecipitation analysis was performed on radiolabeled yeast strains transformed with either myc-tagged YEpSEC9-myc plasmid (pB37) or, as a control, an otherwise identical untagged YEpSEC9 plasmid (pB37) in strains which contained a construct overexpressing either full-length Sro7 (pB363) or CT Sro7 (pB367) under control of the inducible GAL1 promoter. All strains were induced in galactose-containing media for 2 h before 1 h labeling of cells with 35S-Express label in galactose-containing media. The cells were spheroplasted and lysed osmotically in PBS. Lysates were immediately subjected to treatment with the protein cleavable cross-linking agent (DSP) dissolved in DMSO or a DMSO control for 20 min on ice and subsequently boiled in 1% SDS buffer before dilution with IP buffer. Association because of cross-linking was monitored by a two-step immunoprecipitation protocol with the first immunoprecipitation being with the α-myc or directly with α-Sro7p antibodies (α-Sro7 IP). After washing, samples were boiled in 1% SDS/0.1 M DTT (to cleave the cross-linker), diluted with IP buffer, and subjected to a second round of immunoprecipitation with either α-Sro7p (top) or α-Sec9p (bottom) polyclonal antibodies. Samples were boiled in sample buffer, resolved by SDS-PAGE, dried, and exposed to film. The expression from the GAL1-SRO7 and GAL1-SRO7-CT constructs were similar (compare first and last lanes, marked α-Sro7 IP). A cross-linker and myc tag–dependent interaction is apparent between full-length Sro7p and Sec9p, strongly suggesting that these proteins are directly associated with each other in vivo. In contrast, the COOH-terminal domain does not show a detectable cross-linking in this assay.

Mentions: The two-hybrid interaction, post-Golgi secretion defect, and subcellular localization data strongly suggest that Sro7p function is likely to involve it binding to Sec9p in vivo. To test this directly, we examined whether we could coprecipitate Sec9 and Sro7 proteins after treatment with the chemical cross-linker, DSP. Yeast strains were generated that expressed either full-length Sro7p or the COOH-terminal domain of Sro7p (Fig. 1) behind the GAL1 promoter along with either a myc-tagged Sec9p construct or an untagged Sec9p as a control. Cells were radiolabeled with [35S]methionine/cysteine in selective galactose-containing media (to induce the expression of the full-length or COOH-terminal domain of Sro7p), spheroplasted, and lysed. Lysates were either treated with the cleavable cross-linker, DSP, dissolved in DMSO (+DSP) or mock-treated with DMSO alone (−DSP), boiled in 1% SDS (so that only covalent associations with myc-Sec9p are retained), diluted in buffer, and subjected to immunoprecipitation with the α-myc mAb 9E10. These immunoprecipitates were boiled in buffer containing 1% SDS/0.1 M DTT to cleave the cross-linker, and then subjected to immunoprecipitation with either α-Sro7p or α-Sec9p antibodies to monitor the results of the cross-linking. Fig. 8 shows that full-length Sro7p can be coprecipitated with myc-Sec9p in a manner that is dependent both on the presence of the DSP cross-linker and the epitope tag on Sec9p. Interestingly, this interaction was not observed in the strains expressing only the COOH-terminal half of the Sro7 protein, suggesting that whereas this domain is sufficient for binding in vitro, in vivo the interaction is much more efficient with the full-length Sro7 protein.


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)

Full-length Sro7p, but not the COOH-terminal half of Sro7p, can be directly cross-linked to Sec9p in yeast lysates. Coprecipitation analysis was performed on radiolabeled yeast strains transformed with either myc-tagged YEpSEC9-myc plasmid (pB37) or, as a control, an otherwise identical untagged YEpSEC9 plasmid (pB37) in strains which contained a construct overexpressing either full-length Sro7 (pB363) or CT Sro7 (pB367) under control of the inducible GAL1 promoter. All strains were induced in galactose-containing media for 2 h before 1 h labeling of cells with 35S-Express label in galactose-containing media. The cells were spheroplasted and lysed osmotically in PBS. Lysates were immediately subjected to treatment with the protein cleavable cross-linking agent (DSP) dissolved in DMSO or a DMSO control for 20 min on ice and subsequently boiled in 1% SDS buffer before dilution with IP buffer. Association because of cross-linking was monitored by a two-step immunoprecipitation protocol with the first immunoprecipitation being with the α-myc or directly with α-Sro7p antibodies (α-Sro7 IP). After washing, samples were boiled in 1% SDS/0.1 M DTT (to cleave the cross-linker), diluted with IP buffer, and subjected to a second round of immunoprecipitation with either α-Sro7p (top) or α-Sec9p (bottom) polyclonal antibodies. Samples were boiled in sample buffer, resolved by SDS-PAGE, dried, and exposed to film. The expression from the GAL1-SRO7 and GAL1-SRO7-CT constructs were similar (compare first and last lanes, marked α-Sro7 IP). A cross-linker and myc tag–dependent interaction is apparent between full-length Sro7p and Sec9p, strongly suggesting that these proteins are directly associated with each other in vivo. In contrast, the COOH-terminal domain does not show a detectable cross-linking in this assay.
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Related In: Results  -  Collection

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Figure 8: Full-length Sro7p, but not the COOH-terminal half of Sro7p, can be directly cross-linked to Sec9p in yeast lysates. Coprecipitation analysis was performed on radiolabeled yeast strains transformed with either myc-tagged YEpSEC9-myc plasmid (pB37) or, as a control, an otherwise identical untagged YEpSEC9 plasmid (pB37) in strains which contained a construct overexpressing either full-length Sro7 (pB363) or CT Sro7 (pB367) under control of the inducible GAL1 promoter. All strains were induced in galactose-containing media for 2 h before 1 h labeling of cells with 35S-Express label in galactose-containing media. The cells were spheroplasted and lysed osmotically in PBS. Lysates were immediately subjected to treatment with the protein cleavable cross-linking agent (DSP) dissolved in DMSO or a DMSO control for 20 min on ice and subsequently boiled in 1% SDS buffer before dilution with IP buffer. Association because of cross-linking was monitored by a two-step immunoprecipitation protocol with the first immunoprecipitation being with the α-myc or directly with α-Sro7p antibodies (α-Sro7 IP). After washing, samples were boiled in 1% SDS/0.1 M DTT (to cleave the cross-linker), diluted with IP buffer, and subjected to a second round of immunoprecipitation with either α-Sro7p (top) or α-Sec9p (bottom) polyclonal antibodies. Samples were boiled in sample buffer, resolved by SDS-PAGE, dried, and exposed to film. The expression from the GAL1-SRO7 and GAL1-SRO7-CT constructs were similar (compare first and last lanes, marked α-Sro7 IP). A cross-linker and myc tag–dependent interaction is apparent between full-length Sro7p and Sec9p, strongly suggesting that these proteins are directly associated with each other in vivo. In contrast, the COOH-terminal domain does not show a detectable cross-linking in this assay.
Mentions: The two-hybrid interaction, post-Golgi secretion defect, and subcellular localization data strongly suggest that Sro7p function is likely to involve it binding to Sec9p in vivo. To test this directly, we examined whether we could coprecipitate Sec9 and Sro7 proteins after treatment with the chemical cross-linker, DSP. Yeast strains were generated that expressed either full-length Sro7p or the COOH-terminal domain of Sro7p (Fig. 1) behind the GAL1 promoter along with either a myc-tagged Sec9p construct or an untagged Sec9p as a control. Cells were radiolabeled with [35S]methionine/cysteine in selective galactose-containing media (to induce the expression of the full-length or COOH-terminal domain of Sro7p), spheroplasted, and lysed. Lysates were either treated with the cleavable cross-linker, DSP, dissolved in DMSO (+DSP) or mock-treated with DMSO alone (−DSP), boiled in 1% SDS (so that only covalent associations with myc-Sec9p are retained), diluted in buffer, and subjected to immunoprecipitation with the α-myc mAb 9E10. These immunoprecipitates were boiled in buffer containing 1% SDS/0.1 M DTT to cleave the cross-linker, and then subjected to immunoprecipitation with either α-Sro7p or α-Sec9p antibodies to monitor the results of the cross-linking. Fig. 8 shows that full-length Sro7p can be coprecipitated with myc-Sec9p in a manner that is dependent both on the presence of the DSP cross-linker and the epitope tag on Sec9p. Interestingly, this interaction was not observed in the strains expressing only the COOH-terminal half of the Sro7 protein, suggesting that whereas this domain is sufficient for binding in vitro, in vivo the interaction is much more efficient with the full-length Sro7 protein.

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