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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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Compensatory mutation of SNC2 cannot rescue the sporulation defect of sso1Q224R. Strains HI3 (sso1Δ/sso1Δ) or HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) were transformed with the CEN plasmids expressing the indicated genes and sporulated in liquid culture. Sporulation was assessed by observation in the light microscope and by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain. For HI75, the plasmid carrying the wild-type SSO1 was lost by growth on 5-FOA before cells were assayed.
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fig1: Compensatory mutation of SNC2 cannot rescue the sporulation defect of sso1Q224R. Strains HI3 (sso1Δ/sso1Δ) or HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) were transformed with the CEN plasmids expressing the indicated genes and sporulated in liquid culture. Sporulation was assessed by observation in the light microscope and by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain. For HI75, the plasmid carrying the wild-type SSO1 was lost by growth on 5-FOA before cells were assayed.

Mentions: Prospore membrane formation requires the t-SNAREs Sso1p and Spo20p (Neiman, 1998; Jantti et al., 2002). Though the v-SNAREs Snc1p and Snc2p localize to the prospore membrane (Neiman et al., 2000), direct evidence of their involvement in prospore membrane assembly has not been reported. Compensatory Q/R mutations in the central ionic layer of a t-SNARE and a v-SNARE have been used to demonstrate specific SNARE interactions in vivo (Katz and Brennwald, 2000; Graf et al., 2005). To examine the possible role of the Snc proteins during sporulation, we introduced a plasmid carrying the sso1Q224R allele into strain HI3 (sso1/sso1) alone or in combination with a plasmid carrying the snc2R52Q allele. As expected, the sso1Q224R allele did not rescue the sporulation defect of the sso1Δ (Fig. 1). Neither SNC2 nor snc2R52Q were capable of restoring sporulation in this context (Fig. 1). This failure of snc2R52Q to rescue the sporulation defect raises the possibility that Snc2p does not participate in vesicle fusion at the prospore membrane. We therefore examined whether mutation of the central layer arginine to glutamine in any of the other S. cerevisiae R-SNAREs (Snc1, Ykt6, Sec22, Nyv1) could restore sporulation to the sso1Q224R strain. As with snc2R52Q, none of these mutant SNAREs could compensate for the sso1Q224R mutation (unpublished data).


Binding interactions control SNARE specificity in vivo.

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

Compensatory mutation of SNC2 cannot rescue the sporulation defect of sso1Q224R. Strains HI3 (sso1Δ/sso1Δ) or HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) were transformed with the CEN plasmids expressing the indicated genes and sporulated in liquid culture. Sporulation was assessed by observation in the light microscope and by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain. For HI75, the plasmid carrying the wild-type SSO1 was lost by growth on 5-FOA before cells were assayed.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2600744&req=5

fig1: Compensatory mutation of SNC2 cannot rescue the sporulation defect of sso1Q224R. Strains HI3 (sso1Δ/sso1Δ) or HI75 (sso1Δ/sso1Δ sso2Δ/sso2Δ) were transformed with the CEN plasmids expressing the indicated genes and sporulated in liquid culture. Sporulation was assessed by observation in the light microscope and by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain. For HI75, the plasmid carrying the wild-type SSO1 was lost by growth on 5-FOA before cells were assayed.
Mentions: Prospore membrane formation requires the t-SNAREs Sso1p and Spo20p (Neiman, 1998; Jantti et al., 2002). Though the v-SNAREs Snc1p and Snc2p localize to the prospore membrane (Neiman et al., 2000), direct evidence of their involvement in prospore membrane assembly has not been reported. Compensatory Q/R mutations in the central ionic layer of a t-SNARE and a v-SNARE have been used to demonstrate specific SNARE interactions in vivo (Katz and Brennwald, 2000; Graf et al., 2005). To examine the possible role of the Snc proteins during sporulation, we introduced a plasmid carrying the sso1Q224R allele into strain HI3 (sso1/sso1) alone or in combination with a plasmid carrying the snc2R52Q allele. As expected, the sso1Q224R allele did not rescue the sporulation defect of the sso1Δ (Fig. 1). Neither SNC2 nor snc2R52Q were capable of restoring sporulation in this context (Fig. 1). This failure of snc2R52Q to rescue the sporulation defect raises the possibility that Snc2p does not participate in vesicle fusion at the prospore membrane. We therefore examined whether mutation of the central layer arginine to glutamine in any of the other S. cerevisiae R-SNAREs (Snc1, Ykt6, Sec22, Nyv1) could restore sporulation to the sso1Q224R strain. As with snc2R52Q, none of these mutant SNAREs could compensate for the sso1Q224R mutation (unpublished data).

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