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Ordering the final events in yeast exocytosis.

Grote E, Carr CM, Novick PJ - J. Cell Biol. (2000)

Bottom Line: By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly.In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane.Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

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SNARE complex assembly and secretion in exocyst mutant yeast. (a) Variation in SNARE complex amounts with time at 38°C. Wild-type (NY13) cells grown at 25°C were incubated at 38°C for the indicated times. Coimmunoprecipitation of Ssop with Sncp was quantified as described in the legend to Fig. 1 b. The amount of Ssop bound to Sncp at 25°C was defined as 100%. (b) SNARE complex assembly. sec5-24 (NY402), sec6-4 (NY7), sec8-6 (NY47), sec 10-2 (NY61), and sec15-1 (NY64) cells grown at 25°C were incubated at 38°C for the indicated times. The amount of Ssop bound to Sncp in the wild-type at each time point was defined as 100%. (c) Positive correlation between SNARE complex assembly and the secretion rate. Release of 35S-p150 from cells labeled with [35S]methionine at 38°C was quantified by PhosphorImager as described in the legend to Fig. 1 d. The rate of secretion from 4 to 12 min after [35S]methionine addition is plotted. In all strains tested, the secretion rate was constant after the 4 min required for biosynthesis and transport of 35S-labeled proteins through the secretory pathway. The secretion rate in wild-type cells at 38°C was defined as 100%. The amount of Ssop bound to Sncp after 10 min at 38°C (from b) is also displayed to facilitate comparisons.
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Figure 2: SNARE complex assembly and secretion in exocyst mutant yeast. (a) Variation in SNARE complex amounts with time at 38°C. Wild-type (NY13) cells grown at 25°C were incubated at 38°C for the indicated times. Coimmunoprecipitation of Ssop with Sncp was quantified as described in the legend to Fig. 1 b. The amount of Ssop bound to Sncp at 25°C was defined as 100%. (b) SNARE complex assembly. sec5-24 (NY402), sec6-4 (NY7), sec8-6 (NY47), sec 10-2 (NY61), and sec15-1 (NY64) cells grown at 25°C were incubated at 38°C for the indicated times. The amount of Ssop bound to Sncp in the wild-type at each time point was defined as 100%. (c) Positive correlation between SNARE complex assembly and the secretion rate. Release of 35S-p150 from cells labeled with [35S]methionine at 38°C was quantified by PhosphorImager as described in the legend to Fig. 1 d. The rate of secretion from 4 to 12 min after [35S]methionine addition is plotted. In all strains tested, the secretion rate was constant after the 4 min required for biosynthesis and transport of 35S-labeled proteins through the secretory pathway. The secretion rate in wild-type cells at 38°C was defined as 100%. The amount of Ssop bound to Sncp after 10 min at 38°C (from b) is also displayed to facilitate comparisons.

Mentions: To determine whether the exocyst is required for SNARE complex assembly, binding of Ssop to Sncp was measured in several temperature-sensitive exocyst mutant alleles. There was a clear reduction in the amount of Ssop bound to Sncp after 10 min at 37°C in sec5-24, sec6-4, and sec15-1. However, near wild-type levels of Ssop remained bound to Sncp in sec3-2, sec8-9, and sec10-2. These results suggested that components of the exocyst might function both before and after SNARE complex assembly. To examine this interpretation in more detail, we measured the amount of Ssop bound to Sncp at 3, 10, and 30 min after shifting to 38°C and compared these results with the severity of the secretion block (Fig. 2). A transient twofold increase in the amount of Ssop bound to Sncp was observed in the wild-type strain at the 3-min time point (Fig. 2 a). This temporary increase in the abundance of SNARE complexes may reflect an increase in the secretion rate resulting from the temperature shift. To emphasize the effects of mutations in the exocyst components (rather than general effects of the temperature shift), the data on Ssop binding to Sncp in the exocyst mutant strains are expressed as a percentage of the amount of Ssop bound to Sncp in wild-type cells at each time point (Fig. 2 b). In addition, the amount of SNARE complexes present at the 10-min time point is compared with the secretion rate between 4 and 12 min after the addition of [35S]methionine at 38°C (Fig. 2 c).


Ordering the final events in yeast exocytosis.

Grote E, Carr CM, Novick PJ - J. Cell Biol. (2000)

SNARE complex assembly and secretion in exocyst mutant yeast. (a) Variation in SNARE complex amounts with time at 38°C. Wild-type (NY13) cells grown at 25°C were incubated at 38°C for the indicated times. Coimmunoprecipitation of Ssop with Sncp was quantified as described in the legend to Fig. 1 b. The amount of Ssop bound to Sncp at 25°C was defined as 100%. (b) SNARE complex assembly. sec5-24 (NY402), sec6-4 (NY7), sec8-6 (NY47), sec 10-2 (NY61), and sec15-1 (NY64) cells grown at 25°C were incubated at 38°C for the indicated times. The amount of Ssop bound to Sncp in the wild-type at each time point was defined as 100%. (c) Positive correlation between SNARE complex assembly and the secretion rate. Release of 35S-p150 from cells labeled with [35S]methionine at 38°C was quantified by PhosphorImager as described in the legend to Fig. 1 d. The rate of secretion from 4 to 12 min after [35S]methionine addition is plotted. In all strains tested, the secretion rate was constant after the 4 min required for biosynthesis and transport of 35S-labeled proteins through the secretory pathway. The secretion rate in wild-type cells at 38°C was defined as 100%. The amount of Ssop bound to Sncp after 10 min at 38°C (from b) is also displayed to facilitate comparisons.
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Figure 2: SNARE complex assembly and secretion in exocyst mutant yeast. (a) Variation in SNARE complex amounts with time at 38°C. Wild-type (NY13) cells grown at 25°C were incubated at 38°C for the indicated times. Coimmunoprecipitation of Ssop with Sncp was quantified as described in the legend to Fig. 1 b. The amount of Ssop bound to Sncp at 25°C was defined as 100%. (b) SNARE complex assembly. sec5-24 (NY402), sec6-4 (NY7), sec8-6 (NY47), sec 10-2 (NY61), and sec15-1 (NY64) cells grown at 25°C were incubated at 38°C for the indicated times. The amount of Ssop bound to Sncp in the wild-type at each time point was defined as 100%. (c) Positive correlation between SNARE complex assembly and the secretion rate. Release of 35S-p150 from cells labeled with [35S]methionine at 38°C was quantified by PhosphorImager as described in the legend to Fig. 1 d. The rate of secretion from 4 to 12 min after [35S]methionine addition is plotted. In all strains tested, the secretion rate was constant after the 4 min required for biosynthesis and transport of 35S-labeled proteins through the secretory pathway. The secretion rate in wild-type cells at 38°C was defined as 100%. The amount of Ssop bound to Sncp after 10 min at 38°C (from b) is also displayed to facilitate comparisons.
Mentions: To determine whether the exocyst is required for SNARE complex assembly, binding of Ssop to Sncp was measured in several temperature-sensitive exocyst mutant alleles. There was a clear reduction in the amount of Ssop bound to Sncp after 10 min at 37°C in sec5-24, sec6-4, and sec15-1. However, near wild-type levels of Ssop remained bound to Sncp in sec3-2, sec8-9, and sec10-2. These results suggested that components of the exocyst might function both before and after SNARE complex assembly. To examine this interpretation in more detail, we measured the amount of Ssop bound to Sncp at 3, 10, and 30 min after shifting to 38°C and compared these results with the severity of the secretion block (Fig. 2). A transient twofold increase in the amount of Ssop bound to Sncp was observed in the wild-type strain at the 3-min time point (Fig. 2 a). This temporary increase in the abundance of SNARE complexes may reflect an increase in the secretion rate resulting from the temperature shift. To emphasize the effects of mutations in the exocyst components (rather than general effects of the temperature shift), the data on Ssop binding to Sncp in the exocyst mutant strains are expressed as a percentage of the amount of Ssop bound to Sncp in wild-type cells at each time point (Fig. 2 b). In addition, the amount of SNARE complexes present at the 10-min time point is compared with the secretion rate between 4 and 12 min after the addition of [35S]methionine at 38°C (Fig. 2 c).

Bottom Line: By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly.In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane.Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

Show MeSH
Related in: MedlinePlus