Limits...
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.

Show MeSH

Related in: MedlinePlus

Co-expression of snc2R52Q and a chimeric SPO20 rescues the sporulation defect of sso1Q224R. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) was transformed with plasmids carrying the indicated genes and sporulated in liquid culture. SSO1 alleles were expressed from CEN plasmids; SNC2 and SPO20 alleles were expressed from high copy plasmids. Sporulation was assessed by observation in the light microscope or by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain; percentages represent the average of four experiments. “a”, “b”, and “c” illustrate the arrangement of side chain residues at the ionic layer in the different strains.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Co-expression of snc2R52Q and a chimeric SPO20 rescues the sporulation defect of sso1Q224R. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) was transformed with plasmids carrying the indicated genes and sporulated in liquid culture. SSO1 alleles were expressed from CEN plasmids; SNC2 and SPO20 alleles were expressed from high copy plasmids. Sporulation was assessed by observation in the light microscope or by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain; percentages represent the average of four experiments. “a”, “b”, and “c” illustrate the arrangement of side chain residues at the ionic layer in the different strains.

Mentions: An alternative possibility is the existence of a sporulation-specific protein whose interaction with Sso1 and/or Snc2 is sensitive to these mutations. As Sec9 works with these proteins in vegetative cells and Spo20 replaces it during sporulation, Spo20 would be a candidate for such a factor. To test the possibility that the switch to Spo20 during sporulation is the basis for the sso1Q224X phenotypes, we examined the ability of chimeras in which the helices of Spo20 are replaced with those of Sec9 (PSPS) to rescue the sso1Q224R sporulation defect. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) carrying psso1Q224R was transformed with an empty vector, or one carrying snc2R52Q, as well as high copy plasmids expressing either wild-type SPO20 or the PSPS chimera. Expression of snc2R52Q or SPO20 alone did not increase the frequency of sporulation and, similarly, co-overexpression of snc2R52Q and SPO20 had no effect. Expression of PSPS alone resulted in some increase of sporulation; however, coexpression of both snc2R52Q and the PSPS chimera resulted in sporulation at levels comparable to the same strain carrying SSO1 and SPO20 plasmids (Fig. 4). This result demonstrates that snc2R52Q can contribute to suppression of sso1Q224R, indicating that Snc2 does participate in fusion at the prospore membrane. Moreover, in the context of the rearranged central layer, the partner SNARE for Sso1/Snc2 must contain the Sec9 helical domains for sporulation to occur. Spo20 cannot support membrane assembly under these circumstances. Sensitivity of Spo20-containing complexes to perturbations of the ionic layer may also explain the sporulation-specific nature of other sso1Q224X mutations.


Binding interactions control SNARE specificity in vivo.

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

Co-expression of snc2R52Q and a chimeric SPO20 rescues the sporulation defect of sso1Q224R. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) was transformed with plasmids carrying the indicated genes and sporulated in liquid culture. SSO1 alleles were expressed from CEN plasmids; SNC2 and SPO20 alleles were expressed from high copy plasmids. Sporulation was assessed by observation in the light microscope or by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain; percentages represent the average of four experiments. “a”, “b”, and “c” illustrate the arrangement of side chain residues at the ionic layer in the different strains.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Co-expression of snc2R52Q and a chimeric SPO20 rescues the sporulation defect of sso1Q224R. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) was transformed with plasmids carrying the indicated genes and sporulated in liquid culture. SSO1 alleles were expressed from CEN plasmids; SNC2 and SPO20 alleles were expressed from high copy plasmids. Sporulation was assessed by observation in the light microscope or by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain; percentages represent the average of four experiments. “a”, “b”, and “c” illustrate the arrangement of side chain residues at the ionic layer in the different strains.
Mentions: An alternative possibility is the existence of a sporulation-specific protein whose interaction with Sso1 and/or Snc2 is sensitive to these mutations. As Sec9 works with these proteins in vegetative cells and Spo20 replaces it during sporulation, Spo20 would be a candidate for such a factor. To test the possibility that the switch to Spo20 during sporulation is the basis for the sso1Q224X phenotypes, we examined the ability of chimeras in which the helices of Spo20 are replaced with those of Sec9 (PSPS) to rescue the sso1Q224R sporulation defect. Strain HJ3 (sso1Δ/sso1Δ spo20Δ/spo20Δ) carrying psso1Q224R was transformed with an empty vector, or one carrying snc2R52Q, as well as high copy plasmids expressing either wild-type SPO20 or the PSPS chimera. Expression of snc2R52Q or SPO20 alone did not increase the frequency of sporulation and, similarly, co-overexpression of snc2R52Q and SPO20 had no effect. Expression of PSPS alone resulted in some increase of sporulation; however, coexpression of both snc2R52Q and the PSPS chimera resulted in sporulation at levels comparable to the same strain carrying SSO1 and SPO20 plasmids (Fig. 4). This result demonstrates that snc2R52Q can contribute to suppression of sso1Q224R, indicating that Snc2 does participate in fusion at the prospore membrane. Moreover, in the context of the rearranged central layer, the partner SNARE for Sso1/Snc2 must contain the Sec9 helical domains for sporulation to occur. Spo20 cannot support membrane assembly under these circumstances. Sensitivity of Spo20-containing complexes to perturbations of the ionic layer may also explain the sporulation-specific nature of other sso1Q224X mutations.

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