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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 two interface residues in the Spo20 SNARE helix allows it to function with sso1Q224R. (A) Alignment of the interface residues in the SNARE domains of Spo20 and Sec9. Residues chosen for mutation are in blocks. (B) Sporulation of sso1Q224R snc2R52Q strains expressing different forms of SPO20. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) was transformed with plasmids carrying sso1Q224R and snc2R52Q as well as the indicated form of SPO20. The sso1Q224R allele was expressed from a CEN plasmid; snc2R52Q and the SPO20 alleles were expressed from high copy plasmids. These strains were sporulated and sporulation efficiency measured in the light microscope. At least 500 cells were scored for each strain. Results are the average of three experiments. Error bars indicate one standard deviation.
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fig5: Mutation of two interface residues in the Spo20 SNARE helix allows it to function with sso1Q224R. (A) Alignment of the interface residues in the SNARE domains of Spo20 and Sec9. Residues chosen for mutation are in blocks. (B) Sporulation of sso1Q224R snc2R52Q strains expressing different forms of SPO20. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) was transformed with plasmids carrying sso1Q224R and snc2R52Q as well as the indicated form of SPO20. The sso1Q224R allele was expressed from a CEN plasmid; snc2R52Q and the SPO20 alleles were expressed from high copy plasmids. These strains were sporulated and sporulation efficiency measured in the light microscope. At least 500 cells were scored for each strain. Results are the average of three experiments. Error bars indicate one standard deviation.

Mentions: In vitro, Sso1/Spo20–Snc2 complexes have a lower melting temperature than Sso1/Sec9–Snc2 complexes, suggesting that Spo20 binds less tightly to these other SNAREs than does Sec9 (Liu et al., 2007). Packing interactions between side chains of amino acids located at interfaces on the SNARE helices are likely to determine how tightly the SNAREs in a given complex bind to each other. We aligned the interface residues of Spo20 and Sec9 to look for possible suboptimal residues in Spo20 (Fig. 5). As criteria to identify such residues, we looked for differences in the size and/or chemical properties of the side chains. In the first helix, only two positions looked significantly different, a cysteine at the +3 layer of Spo20 that is leucine in Sec9, and a serine at +5 that is an asparagine in Sec9. In the second helix, four differences of note were found; phenylalanines at the −2 and −1 layers that are threonine and leucine, respectively, in Sec9, an alanine in the +4 layer (leucine in Sec9) and a lysine residue at the +6 position (asparagine in Sec9). These six residues were mutated in pairs in the context of an otherwise wild-type SPO20 sequence. The resulting mutants, SPO20C224L,S231N, SPO20F357L,F361T, and SPO20A378L,K385N were all capable of rescuing the sporulation defect of a spo20 mutant, indicating that the mutants encode functional proteins (unpublished data). They were then tested for their ability to rescue the sporulation defects of HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) expressing the sso1Q224R and snc2R52Q alleles (Fig. 5).


Binding interactions control SNARE specificity in vivo.

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

Mutation of two interface residues in the Spo20 SNARE helix allows it to function with sso1Q224R. (A) Alignment of the interface residues in the SNARE domains of Spo20 and Sec9. Residues chosen for mutation are in blocks. (B) Sporulation of sso1Q224R snc2R52Q strains expressing different forms of SPO20. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) was transformed with plasmids carrying sso1Q224R and snc2R52Q as well as the indicated form of SPO20. The sso1Q224R allele was expressed from a CEN plasmid; snc2R52Q and the SPO20 alleles were expressed from high copy plasmids. These strains were sporulated and sporulation efficiency measured in the light microscope. At least 500 cells were scored for each strain. Results are the average of three experiments. 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

fig5: Mutation of two interface residues in the Spo20 SNARE helix allows it to function with sso1Q224R. (A) Alignment of the interface residues in the SNARE domains of Spo20 and Sec9. Residues chosen for mutation are in blocks. (B) Sporulation of sso1Q224R snc2R52Q strains expressing different forms of SPO20. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) was transformed with plasmids carrying sso1Q224R and snc2R52Q as well as the indicated form of SPO20. The sso1Q224R allele was expressed from a CEN plasmid; snc2R52Q and the SPO20 alleles were expressed from high copy plasmids. These strains were sporulated and sporulation efficiency measured in the light microscope. At least 500 cells were scored for each strain. Results are the average of three experiments. Error bars indicate one standard deviation.
Mentions: In vitro, Sso1/Spo20–Snc2 complexes have a lower melting temperature than Sso1/Sec9–Snc2 complexes, suggesting that Spo20 binds less tightly to these other SNAREs than does Sec9 (Liu et al., 2007). Packing interactions between side chains of amino acids located at interfaces on the SNARE helices are likely to determine how tightly the SNAREs in a given complex bind to each other. We aligned the interface residues of Spo20 and Sec9 to look for possible suboptimal residues in Spo20 (Fig. 5). As criteria to identify such residues, we looked for differences in the size and/or chemical properties of the side chains. In the first helix, only two positions looked significantly different, a cysteine at the +3 layer of Spo20 that is leucine in Sec9, and a serine at +5 that is an asparagine in Sec9. In the second helix, four differences of note were found; phenylalanines at the −2 and −1 layers that are threonine and leucine, respectively, in Sec9, an alanine in the +4 layer (leucine in Sec9) and a lysine residue at the +6 position (asparagine in Sec9). These six residues were mutated in pairs in the context of an otherwise wild-type SPO20 sequence. The resulting mutants, SPO20C224L,S231N, SPO20F357L,F361T, and SPO20A378L,K385N were all capable of rescuing the sporulation defect of a spo20 mutant, indicating that the mutants encode functional proteins (unpublished data). They were then tested for their ability to rescue the sporulation defects of HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) expressing the sso1Q224R and snc2R52Q alleles (Fig. 5).

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