Limits...
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.

Show MeSH

Related in: MedlinePlus

Sro7p is not present on post-Golgi secretory vesicles. Cells from a BY29 (sec1-1, ura3-52) strain were shifted to 37°C in YP medium with 0.1% glucose for 2 h to accumulate post-Golgi vesicles and induce expression of invertase. Spheroplasts were prepared from these cells that were lysed osmotically by gentle resuspension in lysis buffer containing 0.8 M sorbitol. A 100,000 g membrane fraction (P3) enriched in post-Golgi vesicles was prepared from a 10,000 g supernatant fraction by differential centrifugation as described previously (Walworth and Novick 1987). The P3 vesicle fraction was resuspended in lysis buffer and layered onto a 20–40% sorbitol velocity gradient as described previously (Brennwald et al. 1994). The top shows the fractionation profile of latent invertase (a marker for secretory vesicles) compared with the distribution of total protein in the gradient. Invertase activity is expressed as micromoles of glucose per minute per fraction. Protein concentrations were determined according to Bradford 1976. The bottom shows the fractionation of Sro7p compared with Snc1/2, a marker for post-Golgi vesicles (Protopopov et al. 1993). The presence of Sro7p and Snc1/2 in the gradients was determined by immunoblotting followed by quantitation on a STORM PhosphorImager using ImageQuant software. Similar results were obtained using a P3 fraction prepared from a sec6-4 strain.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199738&req=5

Figure 7: Sro7p is not present on post-Golgi secretory vesicles. Cells from a BY29 (sec1-1, ura3-52) strain were shifted to 37°C in YP medium with 0.1% glucose for 2 h to accumulate post-Golgi vesicles and induce expression of invertase. Spheroplasts were prepared from these cells that were lysed osmotically by gentle resuspension in lysis buffer containing 0.8 M sorbitol. A 100,000 g membrane fraction (P3) enriched in post-Golgi vesicles was prepared from a 10,000 g supernatant fraction by differential centrifugation as described previously (Walworth and Novick 1987). The P3 vesicle fraction was resuspended in lysis buffer and layered onto a 20–40% sorbitol velocity gradient as described previously (Brennwald et al. 1994). The top shows the fractionation profile of latent invertase (a marker for secretory vesicles) compared with the distribution of total protein in the gradient. Invertase activity is expressed as micromoles of glucose per minute per fraction. Protein concentrations were determined according to Bradford 1976. The bottom shows the fractionation of Sro7p compared with Snc1/2, a marker for post-Golgi vesicles (Protopopov et al. 1993). The presence of Sro7p and Snc1/2 in the gradients was determined by immunoblotting followed by quantitation on a STORM PhosphorImager using ImageQuant software. Similar results were obtained using a P3 fraction prepared from a sec6-4 strain.

Mentions: To determine whether Sro7p is associated with post-Golgi secretory vesicles, we isolated vesicles accumulated in either of two late acting sec mutants, sec1-1 (Fig. 7) or sec6-4 (not shown). Mutants were shifted to 37°C for 2 h to accumulate vesicles, the cells were lysed, and the vesicle-enriched P3 membrane fraction (Walworth and Novick 1987) was layered onto a velocity gradient. After centrifugation, the gradients were divided into 16 fractions and each fraction was assayed for invertase activity and for the vesicle marker Snc1/2. As expected, both vesicle markers migrated as a major peak in the center of the gradient but no detectable peak of Sro7 was found in this region of the gradient. Instead, the majority of Sro7 in the gradient was found at the top of the gradient, whereas a smaller fraction pelleted at the bottom with the plasma membrane markers (Fig. 7). Identical results were found using vesicles isolated from a sec6-4 strain, with the profile of Sro7p demonstrating no Sro7p in the region of the gradient containing the vesicle peak (not shown). Therefore, like Sec9p, Sro7p does not associate with post-Golgi secretory vesicles.


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 not present on post-Golgi secretory vesicles. Cells from a BY29 (sec1-1, ura3-52) strain were shifted to 37°C in YP medium with 0.1% glucose for 2 h to accumulate post-Golgi vesicles and induce expression of invertase. Spheroplasts were prepared from these cells that were lysed osmotically by gentle resuspension in lysis buffer containing 0.8 M sorbitol. A 100,000 g membrane fraction (P3) enriched in post-Golgi vesicles was prepared from a 10,000 g supernatant fraction by differential centrifugation as described previously (Walworth and Novick 1987). The P3 vesicle fraction was resuspended in lysis buffer and layered onto a 20–40% sorbitol velocity gradient as described previously (Brennwald et al. 1994). The top shows the fractionation profile of latent invertase (a marker for secretory vesicles) compared with the distribution of total protein in the gradient. Invertase activity is expressed as micromoles of glucose per minute per fraction. Protein concentrations were determined according to Bradford 1976. The bottom shows the fractionation of Sro7p compared with Snc1/2, a marker for post-Golgi vesicles (Protopopov et al. 1993). The presence of Sro7p and Snc1/2 in the gradients was determined by immunoblotting followed by quantitation on a STORM PhosphorImager using ImageQuant software. Similar results were obtained using a P3 fraction prepared from a sec6-4 strain.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Sro7p is not present on post-Golgi secretory vesicles. Cells from a BY29 (sec1-1, ura3-52) strain were shifted to 37°C in YP medium with 0.1% glucose for 2 h to accumulate post-Golgi vesicles and induce expression of invertase. Spheroplasts were prepared from these cells that were lysed osmotically by gentle resuspension in lysis buffer containing 0.8 M sorbitol. A 100,000 g membrane fraction (P3) enriched in post-Golgi vesicles was prepared from a 10,000 g supernatant fraction by differential centrifugation as described previously (Walworth and Novick 1987). The P3 vesicle fraction was resuspended in lysis buffer and layered onto a 20–40% sorbitol velocity gradient as described previously (Brennwald et al. 1994). The top shows the fractionation profile of latent invertase (a marker for secretory vesicles) compared with the distribution of total protein in the gradient. Invertase activity is expressed as micromoles of glucose per minute per fraction. Protein concentrations were determined according to Bradford 1976. The bottom shows the fractionation of Sro7p compared with Snc1/2, a marker for post-Golgi vesicles (Protopopov et al. 1993). The presence of Sro7p and Snc1/2 in the gradients was determined by immunoblotting followed by quantitation on a STORM PhosphorImager using ImageQuant software. Similar results were obtained using a P3 fraction prepared from a sec6-4 strain.
Mentions: To determine whether Sro7p is associated with post-Golgi secretory vesicles, we isolated vesicles accumulated in either of two late acting sec mutants, sec1-1 (Fig. 7) or sec6-4 (not shown). Mutants were shifted to 37°C for 2 h to accumulate vesicles, the cells were lysed, and the vesicle-enriched P3 membrane fraction (Walworth and Novick 1987) was layered onto a velocity gradient. After centrifugation, the gradients were divided into 16 fractions and each fraction was assayed for invertase activity and for the vesicle marker Snc1/2. As expected, both vesicle markers migrated as a major peak in the center of the gradient but no detectable peak of Sro7 was found in this region of the gradient. Instead, the majority of Sro7 in the gradient was found at the top of the gradient, whereas a smaller fraction pelleted at the bottom with the plasma membrane markers (Fig. 7). Identical results were found using vesicles isolated from a sec6-4 strain, with the profile of Sro7p demonstrating no Sro7p in the region of the gradient containing the vesicle peak (not shown). Therefore, like Sec9p, Sro7p does not associate with post-Golgi secretory vesicles.

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