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Binding interactions control SNARE specificity in vivo.

Yang HJ, Nakanishi H, Liu S, McNew JA, Neiman AM - J. Cell Biol. (2008)

Bottom Line: Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth.Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells.These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA.

ABSTRACT
Saccharomyces cerevisiae contains two SNAP25 paralogues, Sec9 and Spo20, which mediate vesicle fusion at the plasma membrane and the prospore membrane, respectively. Fusion at the prospore membrane is sensitive to perturbation of the central ionic layer of the SNARE complex. Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth. Suppression of the sporulation defect of an sso1 mutant requires expression of a chimeric form of Spo20 carrying the SNARE helices of Sec9. Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells. This mutant form of Spo20 displayed enhanced activity in in vitro fusion assays, as well as tighter binding to Sso1 and Snc2. These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.

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Sporulation is sensitive to mutation of Sso1Q224. Strains HI3 (sso1Δ/sso1Δ) (for Sso1Q224R, Q224P, and Q224K) or HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) (all other substitutions) expressing an SSO1 gene with the indicated amino acid substitution from a CEN plasmid were sporulated, and the percentage of sporulation in the culture was measured. At least 500 cells were counted for each strain. Sporulation efficiency is shown relative to HI75 carrying the wild-type SSO1. Error bars indicate one standard deviation.
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fig3: Sporulation is sensitive to mutation of Sso1Q224. Strains HI3 (sso1Δ/sso1Δ) (for Sso1Q224R, Q224P, and Q224K) or HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) (all other substitutions) expressing an SSO1 gene with the indicated amino acid substitution from a CEN plasmid were sporulated, and the percentage of sporulation in the culture was measured. At least 500 cells were counted for each strain. Sporulation efficiency is shown relative to HI75 carrying the wild-type SSO1. Error bars indicate one standard deviation.

Mentions: The strains carrying the various sso1Q224X alleles as their only SSO were then examined for their ability to sporulate. Unlike growth rate, sporulation was sensitive to changes at this position (Fig. 3). All of the mutations caused a reduction in sporulation efficiency, varying from a two- to fourfold to several hundredfold with small or polar amino acids better tolerated than large hydrophobic or positively charged side chains. Substitution of glutamine 224 to lysine, which causes slow vegetative growth, led to a complete loss of sporulation, as did the arginine and proline mutations (examined in the sso1 single mutant strain) that cannot support vegetative growth. Substitution to leucine or tryptophan had no effect on growth rate, yet these mutants displayed strong sporulation defects. Thus, mutations such as sso1Q224W behave as sporulation-specific alleles of SSO1; they are as effective as wild-type in supporting vegetative growth but poorly support sporulation. These results suggest that the inability of snc2R52Q to rescue the sporulation defect of sso1Q224R might be caused by the sensitivity of sporulation to changes in the ionic layer residue of Sso1, rather than an indication that Snc2 does not function at the prospore membrane.


Binding interactions control SNARE specificity in vivo.

Yang HJ, Nakanishi H, Liu S, McNew JA, Neiman AM - J. Cell Biol. (2008)

Sporulation is sensitive to mutation of Sso1Q224. Strains HI3 (sso1Δ/sso1Δ) (for Sso1Q224R, Q224P, and Q224K) or HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) (all other substitutions) expressing an SSO1 gene with the indicated amino acid substitution from a CEN plasmid were sporulated, and the percentage of sporulation in the culture was measured. At least 500 cells were counted for each strain. Sporulation efficiency is shown relative to HI75 carrying the wild-type SSO1. Error bars indicate one standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600744&req=5

fig3: Sporulation is sensitive to mutation of Sso1Q224. Strains HI3 (sso1Δ/sso1Δ) (for Sso1Q224R, Q224P, and Q224K) or HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) (all other substitutions) expressing an SSO1 gene with the indicated amino acid substitution from a CEN plasmid were sporulated, and the percentage of sporulation in the culture was measured. At least 500 cells were counted for each strain. Sporulation efficiency is shown relative to HI75 carrying the wild-type SSO1. Error bars indicate one standard deviation.
Mentions: The strains carrying the various sso1Q224X alleles as their only SSO were then examined for their ability to sporulate. Unlike growth rate, sporulation was sensitive to changes at this position (Fig. 3). All of the mutations caused a reduction in sporulation efficiency, varying from a two- to fourfold to several hundredfold with small or polar amino acids better tolerated than large hydrophobic or positively charged side chains. Substitution of glutamine 224 to lysine, which causes slow vegetative growth, led to a complete loss of sporulation, as did the arginine and proline mutations (examined in the sso1 single mutant strain) that cannot support vegetative growth. Substitution to leucine or tryptophan had no effect on growth rate, yet these mutants displayed strong sporulation defects. Thus, mutations such as sso1Q224W behave as sporulation-specific alleles of SSO1; they are as effective as wild-type in supporting vegetative growth but poorly support sporulation. These results suggest that the inability of snc2R52Q to rescue the sporulation defect of sso1Q224R might be caused by the sensitivity of sporulation to changes in the ionic layer residue of Sso1, rather than an indication that Snc2 does not function at the prospore membrane.

Bottom Line: Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth.Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells.These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA.

ABSTRACT
Saccharomyces cerevisiae contains two SNAP25 paralogues, Sec9 and Spo20, which mediate vesicle fusion at the plasma membrane and the prospore membrane, respectively. Fusion at the prospore membrane is sensitive to perturbation of the central ionic layer of the SNARE complex. Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth. Suppression of the sporulation defect of an sso1 mutant requires expression of a chimeric form of Spo20 carrying the SNARE helices of Sec9. Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells. This mutant form of Spo20 displayed enhanced activity in in vitro fusion assays, as well as tighter binding to Sso1 and Snc2. These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.

Show MeSH
Related in: MedlinePlus