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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 sec2 mutant yeast. (a and b) SNARE complex assembly. Wild-type (NY13) and sec2-41 (NY130) yeast were grown to early log phase at 25°C. An aliquot of each strain was shifted to 37°C for 10 min. Ssop coimmunoprecipitating with Sncp from a detergent-solubilized lysate was observed by Western blotting (a) and quantified by densitometry (b). The steady-state amount of SNARE complexes in wild-type cells at 25°C was defined as 100%. (c and d) Secretion rate. Cells were grown at 25°C, pelleted, and resuspended in [35S]methionine labeling medium prewarmed to 37°C. At the indicated times (in minutes), cells were pelleted from an aliquot and media proteins were collected by TCA precipitation. The media proteins and 5% of a total cell lysate from the 20-min time point were run on a 5% polyacrylamide gel and detected by autoradiography (c). A 16-h exposure for the secreted proteins and a 30-min exposure for the total cell lysates are presented. Secretion of the 150-kD protein (marked with an arrow in c) was quantified using a PhosphorImager (d).
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Figure 1: SNARE complex assembly and secretion in sec2 mutant yeast. (a and b) SNARE complex assembly. Wild-type (NY13) and sec2-41 (NY130) yeast were grown to early log phase at 25°C. An aliquot of each strain was shifted to 37°C for 10 min. Ssop coimmunoprecipitating with Sncp from a detergent-solubilized lysate was observed by Western blotting (a) and quantified by densitometry (b). The steady-state amount of SNARE complexes in wild-type cells at 25°C was defined as 100%. (c and d) Secretion rate. Cells were grown at 25°C, pelleted, and resuspended in [35S]methionine labeling medium prewarmed to 37°C. At the indicated times (in minutes), cells were pelleted from an aliquot and media proteins were collected by TCA precipitation. The media proteins and 5% of a total cell lysate from the 20-min time point were run on a 5% polyacrylamide gel and detected by autoradiography (c). A 16-h exposure for the secreted proteins and a 30-min exposure for the total cell lysates are presented. Secretion of the 150-kD protein (marked with an arrow in c) was quantified using a PhosphorImager (d).

Mentions: We have previously reported that exocytic SNARE complexes do not assemble at 37°C in sec4-8 mutant yeast. Therefore, we proposed that the Sec4p Rab-GTPase acts upstream of SNARE complex assembly (Grote and Novick 1999). To determine whether Sec4p must be in its active, GTP-bound conformation to promote SNARE complex assembly, we measured the binding of Ssop to Sncp in the sec2-41 mutant. In this strain, deletion of a COOH-terminal targeting domain from Sec2p results in Sec2p mislocalization, and thereby prevents Sec2p from activating Sec4p (Nair et al. 1990; Walch-Solimena et al. 1997; Elkind et al. 2000). An advantage of the sec2-41 strain is that, unlike sec4-8, the cells divide at the same rate as wild-type cells and have wild-type levels of SNARE complexes at 25°C. After shifting sec2-41 cells to 37°C for 10 min, there was an 80% reduction in the amount of Ssop coprecipitated with Sncp compared with wild-type cells at 37°C or sec2-41 cells at 25°C (Fig. 1, a and b). This observation suggests that Sec2p acts upstream of SNARE complex assembly.


Ordering the final events in yeast exocytosis.

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

SNARE complex assembly and secretion in sec2 mutant yeast. (a and b) SNARE complex assembly. Wild-type (NY13) and sec2-41 (NY130) yeast were grown to early log phase at 25°C. An aliquot of each strain was shifted to 37°C for 10 min. Ssop coimmunoprecipitating with Sncp from a detergent-solubilized lysate was observed by Western blotting (a) and quantified by densitometry (b). The steady-state amount of SNARE complexes in wild-type cells at 25°C was defined as 100%. (c and d) Secretion rate. Cells were grown at 25°C, pelleted, and resuspended in [35S]methionine labeling medium prewarmed to 37°C. At the indicated times (in minutes), cells were pelleted from an aliquot and media proteins were collected by TCA precipitation. The media proteins and 5% of a total cell lysate from the 20-min time point were run on a 5% polyacrylamide gel and detected by autoradiography (c). A 16-h exposure for the secreted proteins and a 30-min exposure for the total cell lysates are presented. Secretion of the 150-kD protein (marked with an arrow in c) was quantified using a PhosphorImager (d).
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Figure 1: SNARE complex assembly and secretion in sec2 mutant yeast. (a and b) SNARE complex assembly. Wild-type (NY13) and sec2-41 (NY130) yeast were grown to early log phase at 25°C. An aliquot of each strain was shifted to 37°C for 10 min. Ssop coimmunoprecipitating with Sncp from a detergent-solubilized lysate was observed by Western blotting (a) and quantified by densitometry (b). The steady-state amount of SNARE complexes in wild-type cells at 25°C was defined as 100%. (c and d) Secretion rate. Cells were grown at 25°C, pelleted, and resuspended in [35S]methionine labeling medium prewarmed to 37°C. At the indicated times (in minutes), cells were pelleted from an aliquot and media proteins were collected by TCA precipitation. The media proteins and 5% of a total cell lysate from the 20-min time point were run on a 5% polyacrylamide gel and detected by autoradiography (c). A 16-h exposure for the secreted proteins and a 30-min exposure for the total cell lysates are presented. Secretion of the 150-kD protein (marked with an arrow in c) was quantified using a PhosphorImager (d).
Mentions: We have previously reported that exocytic SNARE complexes do not assemble at 37°C in sec4-8 mutant yeast. Therefore, we proposed that the Sec4p Rab-GTPase acts upstream of SNARE complex assembly (Grote and Novick 1999). To determine whether Sec4p must be in its active, GTP-bound conformation to promote SNARE complex assembly, we measured the binding of Ssop to Sncp in the sec2-41 mutant. In this strain, deletion of a COOH-terminal targeting domain from Sec2p results in Sec2p mislocalization, and thereby prevents Sec2p from activating Sec4p (Nair et al. 1990; Walch-Solimena et al. 1997; Elkind et al. 2000). An advantage of the sec2-41 strain is that, unlike sec4-8, the cells divide at the same rate as wild-type cells and have wild-type levels of SNARE complexes at 25°C. After shifting sec2-41 cells to 37°C for 10 min, there was an 80% reduction in the amount of Ssop coprecipitated with Sncp compared with wild-type cells at 37°C or sec2-41 cells at 25°C (Fig. 1, a and b). This observation suggests that Sec2p acts upstream of SNARE complex assembly.

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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Related in: MedlinePlus