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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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Mutation of Sso1Q224 is well tolerated in vegetative growth. Strain HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) was transformed with CEN plasmids carrying all possible amino acid substitutions at position 224 of Sso1 and the plasmid carrying the wild-type SSO1 was lost by plasmid shuffle, leaving the mutant as the sole form of Sso protein in the cell. Transformants carrying substitutions that could support growth (all except arginine and proline) were streaked out on YPD plates and incubated at 37°C. Letters indicate the amino acid present at position at 224 of Sso1 in each strain.
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fig2: Mutation of Sso1Q224 is well tolerated in vegetative growth. Strain HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) was transformed with CEN plasmids carrying all possible amino acid substitutions at position 224 of Sso1 and the plasmid carrying the wild-type SSO1 was lost by plasmid shuffle, leaving the mutant as the sole form of Sso protein in the cell. Transformants carrying substitutions that could support growth (all except arginine and proline) were streaked out on YPD plates and incubated at 37°C. Letters indicate the amino acid present at position at 224 of Sso1 in each strain.

Mentions: To further explore the role of the central ionic layer in Sso1 function, codon 224 was mutated and alleles bearing all possible amino acid replacements of the glutamine were constructed. Each of these sso1Q224X alleles was introduced on a plasmid into strain HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ pSSO1) and the transformants were then transferred to plates containing 5-fluoroorotic acid (5-FOA) to select for loss of the wild-type SSO1-containing plasmid. Like sso1Q224R, sso1Q224P failed to grow on 5-FOA, indicating that a proline substitution at this position also interferes with Sso1 function. Though arginine cannot function, lysine is weakly tolerated at this position as cells containing sso1Q224K as their only source of Sso protein were viable, but slow growing at elevated temperature (Fig. 2). The phenotype of sso1Q224K may be due to the presence of the positively charged side chain because, as with sso1Q224R, coexpression of snc2R52Q suppressed the slow growth of sso1Q224K (unpublished data). However, other than these three mutations, all other amino acids at position 224 were well tolerated. As judged by colony size, strains carrying these sso1Q224X alleles as their sole form SSO grew as well as those carrying SSO1 even at low or high temperatures (Fig. 2; not depicted). Thus, despite the strong evolutionary conservation of ionic layer glutamine, the yeast plasma membrane SNARE can tolerate a wide variety of residues at this position.


Binding interactions control SNARE specificity in vivo.

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

Mutation of Sso1Q224 is well tolerated in vegetative growth. Strain HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) was transformed with CEN plasmids carrying all possible amino acid substitutions at position 224 of Sso1 and the plasmid carrying the wild-type SSO1 was lost by plasmid shuffle, leaving the mutant as the sole form of Sso protein in the cell. Transformants carrying substitutions that could support growth (all except arginine and proline) were streaked out on YPD plates and incubated at 37°C. Letters indicate the amino acid present at position at 224 of Sso1 in each strain.
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Related In: Results  -  Collection

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fig2: Mutation of Sso1Q224 is well tolerated in vegetative growth. Strain HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) was transformed with CEN plasmids carrying all possible amino acid substitutions at position 224 of Sso1 and the plasmid carrying the wild-type SSO1 was lost by plasmid shuffle, leaving the mutant as the sole form of Sso protein in the cell. Transformants carrying substitutions that could support growth (all except arginine and proline) were streaked out on YPD plates and incubated at 37°C. Letters indicate the amino acid present at position at 224 of Sso1 in each strain.
Mentions: To further explore the role of the central ionic layer in Sso1 function, codon 224 was mutated and alleles bearing all possible amino acid replacements of the glutamine were constructed. Each of these sso1Q224X alleles was introduced on a plasmid into strain HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ pSSO1) and the transformants were then transferred to plates containing 5-fluoroorotic acid (5-FOA) to select for loss of the wild-type SSO1-containing plasmid. Like sso1Q224R, sso1Q224P failed to grow on 5-FOA, indicating that a proline substitution at this position also interferes with Sso1 function. Though arginine cannot function, lysine is weakly tolerated at this position as cells containing sso1Q224K as their only source of Sso protein were viable, but slow growing at elevated temperature (Fig. 2). The phenotype of sso1Q224K may be due to the presence of the positively charged side chain because, as with sso1Q224R, coexpression of snc2R52Q suppressed the slow growth of sso1Q224K (unpublished data). However, other than these three mutations, all other amino acids at position 224 were well tolerated. As judged by colony size, strains carrying these sso1Q224X alleles as their sole form SSO grew as well as those carrying SSO1 even at low or high temperatures (Fig. 2; not depicted). Thus, despite the strong evolutionary conservation of ionic layer glutamine, the yeast plasma membrane SNARE can tolerate a wide variety of residues at this position.

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