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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 accumulation and secretion in sec18-1 mutant yeast. (a) Kinetics of SNARE complex accumulation. Wild-type (NY13) and sec18-1 (NY431) cells were grown at 25°C. The amount of Ssop bound to Sncp was quantified at the indicated times after shifting to 37°C as in the legend to Fig. 1 b. (b) Onset of the secretion block at 37°C. Cells were labeled with [35S]methionine for 5 min at 25°C, pelleted, and resuspended in 37°C chase medium. Aliquots were collected at the indicated times, and secreted 35S-p150 was quantified as in the legend to Fig. 1 d.
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Figure 6: SNARE complex accumulation and secretion in sec18-1 mutant yeast. (a) Kinetics of SNARE complex accumulation. Wild-type (NY13) and sec18-1 (NY431) cells were grown at 25°C. The amount of Ssop bound to Sncp was quantified at the indicated times after shifting to 37°C as in the legend to Fig. 1 b. (b) Onset of the secretion block at 37°C. Cells were labeled with [35S]methionine for 5 min at 25°C, pelleted, and resuspended in 37°C chase medium. Aliquots were collected at the indicated times, and secreted 35S-p150 was quantified as in the legend to Fig. 1 d.

Mentions: To examine the role of Sec18p in yeast exocytic fusion, Ssop binding to Sncp was measured in a sec18-1 mutant strain. The mutant Sec18-1p has a glutamate to glutamine substitution that inhibits ATP hydrolysis by the D1 ATPase domain and consequently inhibits SNARE complex disassembly (Steel et al. 1999). A threefold increase was observed in the amount of Ssop bound to Sncp within 30 s after shifting sec18-1 cells to 37°C (Fig. 6 a). Importantly, in contrast to the transient increase and later decline in SNARE complex levels observed in wild-type cells, there was no decrease in SNARE complex levels at later time points in the sec18-1 mutant, consistent with the biochemical evidence for a defect in SNARE complex disassembly. The observation that there is no further increase in SNARE complex assembly after the first 30 s at 37°C is consistent with the possibility that SNARE proteins must be primed by Sec18p for assembly into trans-SNARE complexes (Ungermann et al. 1998a). However, further SNARE complex assembly is also expected to be inhibited in the sec18-1 mutant as a consequence of the block in transit through the Golgi complex (Grote and Novick 1999).


Ordering the final events in yeast exocytosis.

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

SNARE complex accumulation and secretion in sec18-1 mutant yeast. (a) Kinetics of SNARE complex accumulation. Wild-type (NY13) and sec18-1 (NY431) cells were grown at 25°C. The amount of Ssop bound to Sncp was quantified at the indicated times after shifting to 37°C as in the legend to Fig. 1 b. (b) Onset of the secretion block at 37°C. Cells were labeled with [35S]methionine for 5 min at 25°C, pelleted, and resuspended in 37°C chase medium. Aliquots were collected at the indicated times, and secreted 35S-p150 was quantified as in the legend to Fig. 1 d.
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Related In: Results  -  Collection

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Figure 6: SNARE complex accumulation and secretion in sec18-1 mutant yeast. (a) Kinetics of SNARE complex accumulation. Wild-type (NY13) and sec18-1 (NY431) cells were grown at 25°C. The amount of Ssop bound to Sncp was quantified at the indicated times after shifting to 37°C as in the legend to Fig. 1 b. (b) Onset of the secretion block at 37°C. Cells were labeled with [35S]methionine for 5 min at 25°C, pelleted, and resuspended in 37°C chase medium. Aliquots were collected at the indicated times, and secreted 35S-p150 was quantified as in the legend to Fig. 1 d.
Mentions: To examine the role of Sec18p in yeast exocytic fusion, Ssop binding to Sncp was measured in a sec18-1 mutant strain. The mutant Sec18-1p has a glutamate to glutamine substitution that inhibits ATP hydrolysis by the D1 ATPase domain and consequently inhibits SNARE complex disassembly (Steel et al. 1999). A threefold increase was observed in the amount of Ssop bound to Sncp within 30 s after shifting sec18-1 cells to 37°C (Fig. 6 a). Importantly, in contrast to the transient increase and later decline in SNARE complex levels observed in wild-type cells, there was no decrease in SNARE complex levels at later time points in the sec18-1 mutant, consistent with the biochemical evidence for a defect in SNARE complex disassembly. The observation that there is no further increase in SNARE complex assembly after the first 30 s at 37°C is consistent with the possibility that SNARE proteins must be primed by Sec18p for assembly into trans-SNARE complexes (Ungermann et al. 1998a). However, further SNARE complex assembly is also expected to be inhibited in the sec18-1 mutant as a consequence of the block in transit through the Golgi complex (Grote and Novick 1999).

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