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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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Localization of GFP-Sec1p in sec mutants. (a) Fluorescent images of GFP-Sec1p localization in wild-type (SEC+, NY1696), sec2-41 (NY2222), sec3-2 (NY2214), sec 5-24 (NY2215) sec6-4 (NY2216), sec8-6 (NY2217), sec8-9 (NY2218), sec10-2 (NY2219), and sec15-1 (NY2220) cells at 25°C and after a 10-min incubation at 37°C. Cells were fixed and viewed by epifluorescence microscopy. (b) Quantitation of GFP-Sec1p localization in wild-type and sec mutant cells. The average of duplicate experiments (except for wild-type, which was done in triplicate) is followed by a range that reflects the variability between experiments performed on different days. At 25°C, the percentage of cells with polarized GFP-Sec1 was as follows: wild-type, 39 ± 13% (n = 826); sec2-41, 30 ± 14% (n = 569); sec3-2, 14 ± 6% (n = 753); sec5-24, 52 ± 9% (n = 409); sec6-4, 44 ± 9% (n = 232); sec8-6, 39 ± 9% (n = 708); sec8-9, 40 ± 10% (n = 410); sec10-2, 38 ± 7% (n = 396); and sec15-1, 18 ± 10% (n = 351). At 37°C, the percentage of cells with polarized GFP-Sec1 was as follows: SEC+, 18 ± 5% (n = 1,046); sec2-41, 8 ± 7% (n = 722); sec3-2, 10 ± 8% (n = 945); sec5-24, 6 ± 0.15% (n = 462); sec6-4, 0.8 ± 0.25% (n = 403); sec8-6, 8 ± 4% (n = 816); sec8-9, 3 ± 2% (n = 419); sec10-2, 16 ± 8% (n = 307); and sec15-1, 0 (n = 554).
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Figure 5: Localization of GFP-Sec1p in sec mutants. (a) Fluorescent images of GFP-Sec1p localization in wild-type (SEC+, NY1696), sec2-41 (NY2222), sec3-2 (NY2214), sec 5-24 (NY2215) sec6-4 (NY2216), sec8-6 (NY2217), sec8-9 (NY2218), sec10-2 (NY2219), and sec15-1 (NY2220) cells at 25°C and after a 10-min incubation at 37°C. Cells were fixed and viewed by epifluorescence microscopy. (b) Quantitation of GFP-Sec1p localization in wild-type and sec mutant cells. The average of duplicate experiments (except for wild-type, which was done in triplicate) is followed by a range that reflects the variability between experiments performed on different days. At 25°C, the percentage of cells with polarized GFP-Sec1 was as follows: wild-type, 39 ± 13% (n = 826); sec2-41, 30 ± 14% (n = 569); sec3-2, 14 ± 6% (n = 753); sec5-24, 52 ± 9% (n = 409); sec6-4, 44 ± 9% (n = 232); sec8-6, 39 ± 9% (n = 708); sec8-9, 40 ± 10% (n = 410); sec10-2, 38 ± 7% (n = 396); and sec15-1, 18 ± 10% (n = 351). At 37°C, the percentage of cells with polarized GFP-Sec1 was as follows: SEC+, 18 ± 5% (n = 1,046); sec2-41, 8 ± 7% (n = 722); sec3-2, 10 ± 8% (n = 945); sec5-24, 6 ± 0.15% (n = 462); sec6-4, 0.8 ± 0.25% (n = 403); sec8-6, 8 ± 4% (n = 816); sec8-9, 3 ± 2% (n = 419); sec10-2, 16 ± 8% (n = 307); and sec15-1, 0 (n = 554).

Mentions: We also previously reported a positive correlation between SNARE complex abundance and the concentration of GFP-Sec1p at exocytic sites (Carr et al. 1999). Here, we examine the localization of GFP-Sec1p in additional sec mutants (Fig. 5). In wild-type cells, GFP-Sec1p is concentrated at the tips of newly emerging buds and at mother–daughter necks in cells undergoing cytokinesis (Fig. 5 a); polarized GFP-Sec1p localization is not detected in cells in the isotropic growth phase. To quantify the fluorescence results, the percentage of cells with GFP-Sec1p fluorescence at bud tips or mother–daughter necks was calculated (Fig. 5 b). The percentages shown are based on detectable polarized localization of GFP-Sec1p. Thus, they do not reflect differences in the intensity of polarized fluorescence, which are difficult to quantify (compare wild-type and sec3-2 cells at 25°C; both are images scored positive for localization). At 25°C, 30–50% of wild-type cells displayed polarized localization of GFP-Sec1p. After a 10-min shift to 37°C, localization is detected in ∼20% of the cells, and the intensity is slightly diminished. GFP-Sec1p has a polarized distribution at 25°C in all of the sec mutants except sec15-1 and sec3-2. The reduced localization in sec15-1 at 25°C correlates with a slight reduction in the number of SNARE complexes under these conditions and may simply reflect the higher threshold for detection of the GFP-Sec1p signal. In sec3-2, GFP-Sec1p is present at bud tips and necks, but is also observed in a diffuse distribution throughout the entire cell. Since there is normal expression of GFP-Sec1p and a wild-type level of SNARE complexes in sec3-2, this results suggests that Sec3p may be required for the concentration of SNARE complexes at exocytic sites. However, the lack of polarized GFP-Sec1p localization may also be explained by a negative genetic interaction between the sec3-2 and GFP-SEC1 alleles (see Materials and Methods). In the remainder of the sec mutants, the extent of polarized GFP-Sec1p localization generally correlates with the abundance of assembled SNARE complexes. Accordingly, after 10 min at 37°C, GFP-Sec1p remains polarized in sec10-2, but is depolarized in the vesicle transport mutant sec2-41 and in the remaining exocyst mutants: sec5-24, sec6-4, sec8-6, and sec8-9. We conclude from these results that SNARE complex assembly is required for polarized localization of GFP-Sec1p.


Ordering the final events in yeast exocytosis.

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

Localization of GFP-Sec1p in sec mutants. (a) Fluorescent images of GFP-Sec1p localization in wild-type (SEC+, NY1696), sec2-41 (NY2222), sec3-2 (NY2214), sec 5-24 (NY2215) sec6-4 (NY2216), sec8-6 (NY2217), sec8-9 (NY2218), sec10-2 (NY2219), and sec15-1 (NY2220) cells at 25°C and after a 10-min incubation at 37°C. Cells were fixed and viewed by epifluorescence microscopy. (b) Quantitation of GFP-Sec1p localization in wild-type and sec mutant cells. The average of duplicate experiments (except for wild-type, which was done in triplicate) is followed by a range that reflects the variability between experiments performed on different days. At 25°C, the percentage of cells with polarized GFP-Sec1 was as follows: wild-type, 39 ± 13% (n = 826); sec2-41, 30 ± 14% (n = 569); sec3-2, 14 ± 6% (n = 753); sec5-24, 52 ± 9% (n = 409); sec6-4, 44 ± 9% (n = 232); sec8-6, 39 ± 9% (n = 708); sec8-9, 40 ± 10% (n = 410); sec10-2, 38 ± 7% (n = 396); and sec15-1, 18 ± 10% (n = 351). At 37°C, the percentage of cells with polarized GFP-Sec1 was as follows: SEC+, 18 ± 5% (n = 1,046); sec2-41, 8 ± 7% (n = 722); sec3-2, 10 ± 8% (n = 945); sec5-24, 6 ± 0.15% (n = 462); sec6-4, 0.8 ± 0.25% (n = 403); sec8-6, 8 ± 4% (n = 816); sec8-9, 3 ± 2% (n = 419); sec10-2, 16 ± 8% (n = 307); and sec15-1, 0 (n = 554).
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Figure 5: Localization of GFP-Sec1p in sec mutants. (a) Fluorescent images of GFP-Sec1p localization in wild-type (SEC+, NY1696), sec2-41 (NY2222), sec3-2 (NY2214), sec 5-24 (NY2215) sec6-4 (NY2216), sec8-6 (NY2217), sec8-9 (NY2218), sec10-2 (NY2219), and sec15-1 (NY2220) cells at 25°C and after a 10-min incubation at 37°C. Cells were fixed and viewed by epifluorescence microscopy. (b) Quantitation of GFP-Sec1p localization in wild-type and sec mutant cells. The average of duplicate experiments (except for wild-type, which was done in triplicate) is followed by a range that reflects the variability between experiments performed on different days. At 25°C, the percentage of cells with polarized GFP-Sec1 was as follows: wild-type, 39 ± 13% (n = 826); sec2-41, 30 ± 14% (n = 569); sec3-2, 14 ± 6% (n = 753); sec5-24, 52 ± 9% (n = 409); sec6-4, 44 ± 9% (n = 232); sec8-6, 39 ± 9% (n = 708); sec8-9, 40 ± 10% (n = 410); sec10-2, 38 ± 7% (n = 396); and sec15-1, 18 ± 10% (n = 351). At 37°C, the percentage of cells with polarized GFP-Sec1 was as follows: SEC+, 18 ± 5% (n = 1,046); sec2-41, 8 ± 7% (n = 722); sec3-2, 10 ± 8% (n = 945); sec5-24, 6 ± 0.15% (n = 462); sec6-4, 0.8 ± 0.25% (n = 403); sec8-6, 8 ± 4% (n = 816); sec8-9, 3 ± 2% (n = 419); sec10-2, 16 ± 8% (n = 307); and sec15-1, 0 (n = 554).
Mentions: We also previously reported a positive correlation between SNARE complex abundance and the concentration of GFP-Sec1p at exocytic sites (Carr et al. 1999). Here, we examine the localization of GFP-Sec1p in additional sec mutants (Fig. 5). In wild-type cells, GFP-Sec1p is concentrated at the tips of newly emerging buds and at mother–daughter necks in cells undergoing cytokinesis (Fig. 5 a); polarized GFP-Sec1p localization is not detected in cells in the isotropic growth phase. To quantify the fluorescence results, the percentage of cells with GFP-Sec1p fluorescence at bud tips or mother–daughter necks was calculated (Fig. 5 b). The percentages shown are based on detectable polarized localization of GFP-Sec1p. Thus, they do not reflect differences in the intensity of polarized fluorescence, which are difficult to quantify (compare wild-type and sec3-2 cells at 25°C; both are images scored positive for localization). At 25°C, 30–50% of wild-type cells displayed polarized localization of GFP-Sec1p. After a 10-min shift to 37°C, localization is detected in ∼20% of the cells, and the intensity is slightly diminished. GFP-Sec1p has a polarized distribution at 25°C in all of the sec mutants except sec15-1 and sec3-2. The reduced localization in sec15-1 at 25°C correlates with a slight reduction in the number of SNARE complexes under these conditions and may simply reflect the higher threshold for detection of the GFP-Sec1p signal. In sec3-2, GFP-Sec1p is present at bud tips and necks, but is also observed in a diffuse distribution throughout the entire cell. Since there is normal expression of GFP-Sec1p and a wild-type level of SNARE complexes in sec3-2, this results suggests that Sec3p may be required for the concentration of SNARE complexes at exocytic sites. However, the lack of polarized GFP-Sec1p localization may also be explained by a negative genetic interaction between the sec3-2 and GFP-SEC1 alleles (see Materials and Methods). In the remainder of the sec mutants, the extent of polarized GFP-Sec1p localization generally correlates with the abundance of assembled SNARE complexes. Accordingly, after 10 min at 37°C, GFP-Sec1p remains polarized in sec10-2, but is depolarized in the vesicle transport mutant sec2-41 and in the remaining exocyst mutants: sec5-24, sec6-4, sec8-6, and sec8-9. We conclude from these results that SNARE complex assembly is required for polarized localization of GFP-Sec1p.

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