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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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(A) Loss of Sro7 and Sro77 leads to the accumulation of a large number of post-Golgi secretory vesicles at the nonpermissive temperature. The wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains were grown to mid-log phase at 37°C, shifted to the restrictive temperature of 19°C for 3 h, and then processed for thin section electron microscopy. High magnification micrographs of the accumulated vesicles demonstrated that >90% of the vesicles had diameters between 80 and 100 nm, identical to that reported for other post-Golgi sec mutants (Novick et al. 1980). (B) Actin localization in wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains. Haploid cells were grown overnight at the permissive temperature of 37°C to log phase, and then shifted to the restrictive temperature of 19°C for 3 h before fixing, permeabilizing, and staining with TRITC-phalloidin. Bars: A, 1 μm; B, 5 μm.
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Figure 4: (A) Loss of Sro7 and Sro77 leads to the accumulation of a large number of post-Golgi secretory vesicles at the nonpermissive temperature. The wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains were grown to mid-log phase at 37°C, shifted to the restrictive temperature of 19°C for 3 h, and then processed for thin section electron microscopy. High magnification micrographs of the accumulated vesicles demonstrated that >90% of the vesicles had diameters between 80 and 100 nm, identical to that reported for other post-Golgi sec mutants (Novick et al. 1980). (B) Actin localization in wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains. Haploid cells were grown overnight at the permissive temperature of 37°C to log phase, and then shifted to the restrictive temperature of 19°C for 3 h before fixing, permeabilizing, and staining with TRITC-phalloidin. Bars: A, 1 μm; B, 5 μm.

Mentions: Another feature common to all known mutants blocked in Golgi-to-cell surface transport in yeast is the accumulation of 80–100 nm secretory vesicles (Novick et al. 1980). To examine the morphological effects of loss of Sro7 and Sro77, we performed thin section electron microscopy (Walworth et al. 1992) on the double-disruptant strain following the same cold temperature shift protocol used in the invertase secretion assays. Representative mutant and wild-type cells are shown in Fig. 4 A. While the mutant cells generally appeared somewhat larger on average than the wild-type cells, the most striking feature was that virtually all of the budded cells observed in the sro7Δ, sro77Δ mutant strain showed a dramatic accumulation of 80–100 nm vesicles. Furthermore, in most of the budded cells the vesicles appeared to be significantly more abundant in the bud than in the mother cell as is the case with most of the late sec mutants (Walch-Solimena et al. 1997). As expected, because of the normally rapid rate of exocytosis in yeast, vesicles were quite rare in wild-type (SRO7, SRO77) cells analyzed in parallel with the mutant strain.


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)

(A) Loss of Sro7 and Sro77 leads to the accumulation of a large number of post-Golgi secretory vesicles at the nonpermissive temperature. The wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains were grown to mid-log phase at 37°C, shifted to the restrictive temperature of 19°C for 3 h, and then processed for thin section electron microscopy. High magnification micrographs of the accumulated vesicles demonstrated that >90% of the vesicles had diameters between 80 and 100 nm, identical to that reported for other post-Golgi sec mutants (Novick et al. 1980). (B) Actin localization in wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains. Haploid cells were grown overnight at the permissive temperature of 37°C to log phase, and then shifted to the restrictive temperature of 19°C for 3 h before fixing, permeabilizing, and staining with TRITC-phalloidin. Bars: A, 1 μm; B, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: (A) Loss of Sro7 and Sro77 leads to the accumulation of a large number of post-Golgi secretory vesicles at the nonpermissive temperature. The wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains were grown to mid-log phase at 37°C, shifted to the restrictive temperature of 19°C for 3 h, and then processed for thin section electron microscopy. High magnification micrographs of the accumulated vesicles demonstrated that >90% of the vesicles had diameters between 80 and 100 nm, identical to that reported for other post-Golgi sec mutants (Novick et al. 1980). (B) Actin localization in wild-type (SRO7, SRO77) and double-disruptant (sro7Δ, sro77Δ) strains. Haploid cells were grown overnight at the permissive temperature of 37°C to log phase, and then shifted to the restrictive temperature of 19°C for 3 h before fixing, permeabilizing, and staining with TRITC-phalloidin. Bars: A, 1 μm; B, 5 μm.
Mentions: Another feature common to all known mutants blocked in Golgi-to-cell surface transport in yeast is the accumulation of 80–100 nm secretory vesicles (Novick et al. 1980). To examine the morphological effects of loss of Sro7 and Sro77, we performed thin section electron microscopy (Walworth et al. 1992) on the double-disruptant strain following the same cold temperature shift protocol used in the invertase secretion assays. Representative mutant and wild-type cells are shown in Fig. 4 A. While the mutant cells generally appeared somewhat larger on average than the wild-type cells, the most striking feature was that virtually all of the budded cells observed in the sro7Δ, sro77Δ mutant strain showed a dramatic accumulation of 80–100 nm vesicles. Furthermore, in most of the budded cells the vesicles appeared to be significantly more abundant in the bud than in the mother cell as is the case with most of the late sec mutants (Walch-Solimena et al. 1997). As expected, because of the normally rapid rate of exocytosis in yeast, vesicles were quite rare in wild-type (SRO7, SRO77) cells analyzed in parallel with the mutant strain.

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