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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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Suppression of the rho3 deletion by multicopy SRO7 and SEC9. A diploid heterozygous for a disruption of the chromosomal rho3 gene (a/α, rho3Δ::LEU2/RHO3, leu2-3/leu2-3; ura3-52/ura3-52) was transformed with multicopy plasmids containing (top) empty vector (pB23), (middle) SRO7 (pB427), or (bottom) SEC9 (pB35). Tetrads were dissected on YPD plates and grown for 3 d at 25°C.
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Figure 11: Suppression of the rho3 deletion by multicopy SRO7 and SEC9. A diploid heterozygous for a disruption of the chromosomal rho3 gene (a/α, rho3Δ::LEU2/RHO3, leu2-3/leu2-3; ura3-52/ura3-52) was transformed with multicopy plasmids containing (top) empty vector (pB23), (middle) SRO7 (pB427), or (bottom) SEC9 (pB35). Tetrads were dissected on YPD plates and grown for 3 d at 25°C.

Mentions: SRO7 was independently isolated as a multicopy suppressor of a mutant in the Rho GTPase, RHO3, which is thought to play a role in regulating actin polarity of the yeast cell. rho3 mutants appear to have severe defects in cytoskeletal organization. Specifically, cortical actin patches, normally present almost exclusively in the bud, are found randomly distributed throughout the mother and bud of the rho3 mutant cells (Imai et al. 1996). Rho3 has also been implicated in polarized exocytosis. This observation stems from the recent identification, by our laboratory, of the RHO3 gene as a high copy suppressor of sec4-P48, a cold-sensitive Sec4 effector domain mutant. Furthermore, we have demonstrated that rho3 mutant cells have a defect in polarity and accumulate post-Golgi secretory vesicles in the mother cell at the restrictive temperature (Adamo, J., G. Rossi, and P. Brennwald, manuscript submitted for publication). Genetic interactions involving RHO3 and SEC4 prompted us to determine if both SRO7 and SEC9 can act as suppressors of a rho3 deletion mutant. Sporulation of a diploid heterozygous for disruption of the chromosomal RHO3 gene results in a 2:2 segregation pattern for growth at 25°C that is linked to the rho3 disruption. Transformation of the high copy vector, pRS426, into the heterozygous diploid did not alter this segregation pattern after sporulation (Fig. 11, top). Expression of high copy SRO7 strongly suppressed the growth defect associated with disruption of rho3 (Fig. 11, middle). SEC9, when expressed on high copy, was even more potent in its suppression of the rho3 deletion (Fig. 11, bottom).


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)

Suppression of the rho3 deletion by multicopy SRO7 and SEC9. A diploid heterozygous for a disruption of the chromosomal rho3 gene (a/α, rho3Δ::LEU2/RHO3, leu2-3/leu2-3; ura3-52/ura3-52) was transformed with multicopy plasmids containing (top) empty vector (pB23), (middle) SRO7 (pB427), or (bottom) SEC9 (pB35). Tetrads were dissected on YPD plates and grown for 3 d at 25°C.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199738&req=5

Figure 11: Suppression of the rho3 deletion by multicopy SRO7 and SEC9. A diploid heterozygous for a disruption of the chromosomal rho3 gene (a/α, rho3Δ::LEU2/RHO3, leu2-3/leu2-3; ura3-52/ura3-52) was transformed with multicopy plasmids containing (top) empty vector (pB23), (middle) SRO7 (pB427), or (bottom) SEC9 (pB35). Tetrads were dissected on YPD plates and grown for 3 d at 25°C.
Mentions: SRO7 was independently isolated as a multicopy suppressor of a mutant in the Rho GTPase, RHO3, which is thought to play a role in regulating actin polarity of the yeast cell. rho3 mutants appear to have severe defects in cytoskeletal organization. Specifically, cortical actin patches, normally present almost exclusively in the bud, are found randomly distributed throughout the mother and bud of the rho3 mutant cells (Imai et al. 1996). Rho3 has also been implicated in polarized exocytosis. This observation stems from the recent identification, by our laboratory, of the RHO3 gene as a high copy suppressor of sec4-P48, a cold-sensitive Sec4 effector domain mutant. Furthermore, we have demonstrated that rho3 mutant cells have a defect in polarity and accumulate post-Golgi secretory vesicles in the mother cell at the restrictive temperature (Adamo, J., G. Rossi, and P. Brennwald, manuscript submitted for publication). Genetic interactions involving RHO3 and SEC4 prompted us to determine if both SRO7 and SEC9 can act as suppressors of a rho3 deletion mutant. Sporulation of a diploid heterozygous for disruption of the chromosomal RHO3 gene results in a 2:2 segregation pattern for growth at 25°C that is linked to the rho3 disruption. Transformation of the high copy vector, pRS426, into the heterozygous diploid did not alter this segregation pattern after sporulation (Fig. 11, top). Expression of high copy SRO7 strongly suppressed the growth defect associated with disruption of rho3 (Fig. 11, middle). SEC9, when expressed on high copy, was even more potent in its suppression of the rho3 deletion (Fig. 11, bottom).

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