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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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The sro7Δ, sro77Δ mutant has a pronounced defect in Golgi-to-cell surface transport. (A) Invertase secretion is deficient in the sro7Δ, sro77Δ mutant strain. Invertase secretion assays were performed after a temperature shift on several tetratype tetrads and a representative example is shown. The left frame shows a comparison of the percentage of invertase secreted into the periplasm after a 3-h shift to the restrictive temperature (19°C). The right frame shows the partitioning of invertase activity in the internal (intracellular) and external (periplasmic) fractions of these cells. All samples were measured in duplicate and the SD determined from two separate experiments. (B) The invertase that accumulates in sro7Δ, sro77Δ cells is fully glycosylated and transport of CPY to the vacuole is unaffected. Wild-type, single-disruptant, and double-disruptant strains, in addition to sec4-8 and sec18-1 strains, were grown and shifted as in A, except that sec4-8 and sec18-1 strains were grown for 2 h at 37°C before lysis. Equivalent amounts of each strain were lysed with glass beads, boiled in SDS sample buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies raised against invertase (top) or CPY (bottom). Mutants that block secretion after exit from the Golgi apparatus, such as sec4-8, accumulate internal pools of fully glycosylated invertase, whereas mutants that block at earlier stages, such as sec18-1, accumulate the core-glycosylated or ER-modified form of invertase. Likewise post-Golgi sec mutants do not affect CPY transport and maturation (and therefore show only the mature, mCPY form), unlike mutants that block earlier in the pathway such as sec18-1, which accumulates the core-glycosylated, p1 form of CPY. The forms of invertase and CPY found in the sro7Δ, sro77Δ cells suggest it is primarily affecting Golgi-to-cell surface transport.
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Figure 3: The sro7Δ, sro77Δ mutant has a pronounced defect in Golgi-to-cell surface transport. (A) Invertase secretion is deficient in the sro7Δ, sro77Δ mutant strain. Invertase secretion assays were performed after a temperature shift on several tetratype tetrads and a representative example is shown. The left frame shows a comparison of the percentage of invertase secreted into the periplasm after a 3-h shift to the restrictive temperature (19°C). The right frame shows the partitioning of invertase activity in the internal (intracellular) and external (periplasmic) fractions of these cells. All samples were measured in duplicate and the SD determined from two separate experiments. (B) The invertase that accumulates in sro7Δ, sro77Δ cells is fully glycosylated and transport of CPY to the vacuole is unaffected. Wild-type, single-disruptant, and double-disruptant strains, in addition to sec4-8 and sec18-1 strains, were grown and shifted as in A, except that sec4-8 and sec18-1 strains were grown for 2 h at 37°C before lysis. Equivalent amounts of each strain were lysed with glass beads, boiled in SDS sample buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies raised against invertase (top) or CPY (bottom). Mutants that block secretion after exit from the Golgi apparatus, such as sec4-8, accumulate internal pools of fully glycosylated invertase, whereas mutants that block at earlier stages, such as sec18-1, accumulate the core-glycosylated or ER-modified form of invertase. Likewise post-Golgi sec mutants do not affect CPY transport and maturation (and therefore show only the mature, mCPY form), unlike mutants that block earlier in the pathway such as sec18-1, which accumulates the core-glycosylated, p1 form of CPY. The forms of invertase and CPY found in the sro7Δ, sro77Δ cells suggest it is primarily affecting Golgi-to-cell surface transport.

Mentions: This conditional growth defect allowed us to assess directly Sro7 and Sro77 function in the yeast secretory pathway. We examined the secretory capacity of sro7Δ, sro77Δ double-disruptant cells after a shift from the permissive temperature of 37°C to the nonpermissive temperature of 19°C. In particular, we determined the ability to secrete invertase (Esmon et al. 1981), a protein that follows the classical secretory pathway from the ER to the extracellular periplasmic space. We performed invertase secretion assays on wild-type (SRO7, SRO77), each of the two single-disruptants (sro7Δ) and (sro77Δ), and the double-disruptant (sro7Δ, sro77Δ) strains. After a 3-h shift to low glucose media (to derepress expression of invertase) at 19°C, the wild-type strain and each of the single disruption strains secreted 88–98% of the total invertase produced in this period. In contrast, the sro7Δ, sro77Δ double mutant strain secreted only 53% of the total invertase produced during the shift, with the remaining 47% being intracellular (Fig. 3 A). These results demonstrate a clear defect in the secretory pathway in response to loss of both Sro7 and Sro77.


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)

The sro7Δ, sro77Δ mutant has a pronounced defect in Golgi-to-cell surface transport. (A) Invertase secretion is deficient in the sro7Δ, sro77Δ mutant strain. Invertase secretion assays were performed after a temperature shift on several tetratype tetrads and a representative example is shown. The left frame shows a comparison of the percentage of invertase secreted into the periplasm after a 3-h shift to the restrictive temperature (19°C). The right frame shows the partitioning of invertase activity in the internal (intracellular) and external (periplasmic) fractions of these cells. All samples were measured in duplicate and the SD determined from two separate experiments. (B) The invertase that accumulates in sro7Δ, sro77Δ cells is fully glycosylated and transport of CPY to the vacuole is unaffected. Wild-type, single-disruptant, and double-disruptant strains, in addition to sec4-8 and sec18-1 strains, were grown and shifted as in A, except that sec4-8 and sec18-1 strains were grown for 2 h at 37°C before lysis. Equivalent amounts of each strain were lysed with glass beads, boiled in SDS sample buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies raised against invertase (top) or CPY (bottom). Mutants that block secretion after exit from the Golgi apparatus, such as sec4-8, accumulate internal pools of fully glycosylated invertase, whereas mutants that block at earlier stages, such as sec18-1, accumulate the core-glycosylated or ER-modified form of invertase. Likewise post-Golgi sec mutants do not affect CPY transport and maturation (and therefore show only the mature, mCPY form), unlike mutants that block earlier in the pathway such as sec18-1, which accumulates the core-glycosylated, p1 form of CPY. The forms of invertase and CPY found in the sro7Δ, sro77Δ cells suggest it is primarily affecting Golgi-to-cell surface transport.
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Related In: Results  -  Collection

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Figure 3: The sro7Δ, sro77Δ mutant has a pronounced defect in Golgi-to-cell surface transport. (A) Invertase secretion is deficient in the sro7Δ, sro77Δ mutant strain. Invertase secretion assays were performed after a temperature shift on several tetratype tetrads and a representative example is shown. The left frame shows a comparison of the percentage of invertase secreted into the periplasm after a 3-h shift to the restrictive temperature (19°C). The right frame shows the partitioning of invertase activity in the internal (intracellular) and external (periplasmic) fractions of these cells. All samples were measured in duplicate and the SD determined from two separate experiments. (B) The invertase that accumulates in sro7Δ, sro77Δ cells is fully glycosylated and transport of CPY to the vacuole is unaffected. Wild-type, single-disruptant, and double-disruptant strains, in addition to sec4-8 and sec18-1 strains, were grown and shifted as in A, except that sec4-8 and sec18-1 strains were grown for 2 h at 37°C before lysis. Equivalent amounts of each strain were lysed with glass beads, boiled in SDS sample buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies raised against invertase (top) or CPY (bottom). Mutants that block secretion after exit from the Golgi apparatus, such as sec4-8, accumulate internal pools of fully glycosylated invertase, whereas mutants that block at earlier stages, such as sec18-1, accumulate the core-glycosylated or ER-modified form of invertase. Likewise post-Golgi sec mutants do not affect CPY transport and maturation (and therefore show only the mature, mCPY form), unlike mutants that block earlier in the pathway such as sec18-1, which accumulates the core-glycosylated, p1 form of CPY. The forms of invertase and CPY found in the sro7Δ, sro77Δ cells suggest it is primarily affecting Golgi-to-cell surface transport.
Mentions: This conditional growth defect allowed us to assess directly Sro7 and Sro77 function in the yeast secretory pathway. We examined the secretory capacity of sro7Δ, sro77Δ double-disruptant cells after a shift from the permissive temperature of 37°C to the nonpermissive temperature of 19°C. In particular, we determined the ability to secrete invertase (Esmon et al. 1981), a protein that follows the classical secretory pathway from the ER to the extracellular periplasmic space. We performed invertase secretion assays on wild-type (SRO7, SRO77), each of the two single-disruptants (sro7Δ) and (sro77Δ), and the double-disruptant (sro7Δ, sro77Δ) strains. After a 3-h shift to low glucose media (to derepress expression of invertase) at 19°C, the wild-type strain and each of the single disruption strains secreted 88–98% of the total invertase produced in this period. In contrast, the sro7Δ, sro77Δ double mutant strain secreted only 53% of the total invertase produced during the shift, with the remaining 47% being intracellular (Fig. 3 A). These results demonstrate a clear defect in the secretory pathway in response to loss of both Sro7 and Sro77.

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