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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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SPO20C224L,S231N can rescue the growth defect of sec9-4. (A) Strain AN211 (sec9-4) was transformed with integrating or centromeric (for PSPP and PPPS) plasmids expressing the indicated genes. Cells were grown to saturation in rich medium and 10-fold serial dilutions were spotted onto selective plates incubated at permissive (25°C) or restrictive (37°C) temperature. SPO20CS,LN is SPO20C224L,S231N; SPO20CSFF,LNTL is SPO20C224L,S231N,F357T,F361L; SPO20CSFFAK,LNTLLN is SPO20C224L,S231N,F357T,F361L,A378K,K385N. (B) A similar growth assay in strain AN211 (sec9-4) using high copy plasmids to express the indicated genes.
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fig6: SPO20C224L,S231N can rescue the growth defect of sec9-4. (A) Strain AN211 (sec9-4) was transformed with integrating or centromeric (for PSPP and PPPS) plasmids expressing the indicated genes. Cells were grown to saturation in rich medium and 10-fold serial dilutions were spotted onto selective plates incubated at permissive (25°C) or restrictive (37°C) temperature. SPO20CS,LN is SPO20C224L,S231N; SPO20CSFF,LNTL is SPO20C224L,S231N,F357T,F361L; SPO20CSFFAK,LNTLLN is SPO20C224L,S231N,F357T,F361L,A378K,K385N. (B) A similar growth assay in strain AN211 (sec9-4) using high copy plasmids to express the indicated genes.

Mentions: Ectopic expression of SPO20 cannot rescue the temperature-sensitive growth defect of a sec9-4 mutant, though a chimeric form of Spo20 carrying the Sec9 helical regions can rescue sec9-4 (Neiman et al., 2000). This suggests that the inability of Spo20 to function at the plasma membrane is tied directly to its SNARE domain. We examined whether the SPO20C224L,S231N allele affects the ability of Spo20 to compensate for loss of SEC9. These experiments were performed with proteins lacking the inhibitory domain (amino acids 3–51) present in the N terminus of Spo20 (Neiman et al., 2000). As previously reported, Δ3-51SPO20 cannot rescue sec9-4, even when present on a high copy plasmid, though Δ3-51PSPS was capable of rescuing growth at high temperature. The Δ3-51SPO20C224L,S231N allele also rescued growth of this strain at 37°C when present in high copy, though neither SPO20C224L,S231N nor the PSPP chimera could rescue when expressed from centromeric plasmids (Fig. 6). These results again suggest that the SPO20C224L,S231N mutations increase the strength of Spo20/Sso/Snc interactions, though not to the same degree as complete replacement with the Sec9 helices. To ensure the differences seen were not due to differential stability of the proteins, 3xHA-tagged versions of the different SPO20 and chimera genes were constructed. Western blotting with anti-HA antibodies indicated that all the SPO20 forms were present in comparable amounts (unpublished data). Therefore, the results reflect differences in the ability of the different forms to promote vesicle fusion, not differences in protein stability.


Binding interactions control SNARE specificity in vivo.

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

SPO20C224L,S231N can rescue the growth defect of sec9-4. (A) Strain AN211 (sec9-4) was transformed with integrating or centromeric (for PSPP and PPPS) plasmids expressing the indicated genes. Cells were grown to saturation in rich medium and 10-fold serial dilutions were spotted onto selective plates incubated at permissive (25°C) or restrictive (37°C) temperature. SPO20CS,LN is SPO20C224L,S231N; SPO20CSFF,LNTL is SPO20C224L,S231N,F357T,F361L; SPO20CSFFAK,LNTLLN is SPO20C224L,S231N,F357T,F361L,A378K,K385N. (B) A similar growth assay in strain AN211 (sec9-4) using high copy plasmids to express the indicated genes.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: SPO20C224L,S231N can rescue the growth defect of sec9-4. (A) Strain AN211 (sec9-4) was transformed with integrating or centromeric (for PSPP and PPPS) plasmids expressing the indicated genes. Cells were grown to saturation in rich medium and 10-fold serial dilutions were spotted onto selective plates incubated at permissive (25°C) or restrictive (37°C) temperature. SPO20CS,LN is SPO20C224L,S231N; SPO20CSFF,LNTL is SPO20C224L,S231N,F357T,F361L; SPO20CSFFAK,LNTLLN is SPO20C224L,S231N,F357T,F361L,A378K,K385N. (B) A similar growth assay in strain AN211 (sec9-4) using high copy plasmids to express the indicated genes.
Mentions: Ectopic expression of SPO20 cannot rescue the temperature-sensitive growth defect of a sec9-4 mutant, though a chimeric form of Spo20 carrying the Sec9 helical regions can rescue sec9-4 (Neiman et al., 2000). This suggests that the inability of Spo20 to function at the plasma membrane is tied directly to its SNARE domain. We examined whether the SPO20C224L,S231N allele affects the ability of Spo20 to compensate for loss of SEC9. These experiments were performed with proteins lacking the inhibitory domain (amino acids 3–51) present in the N terminus of Spo20 (Neiman et al., 2000). As previously reported, Δ3-51SPO20 cannot rescue sec9-4, even when present on a high copy plasmid, though Δ3-51PSPS was capable of rescuing growth at high temperature. The Δ3-51SPO20C224L,S231N allele also rescued growth of this strain at 37°C when present in high copy, though neither SPO20C224L,S231N nor the PSPP chimera could rescue when expressed from centromeric plasmids (Fig. 6). These results again suggest that the SPO20C224L,S231N mutations increase the strength of Spo20/Sso/Snc interactions, though not to the same degree as complete replacement with the Sec9 helices. To ensure the differences seen were not due to differential stability of the proteins, 3xHA-tagged versions of the different SPO20 and chimera genes were constructed. Western blotting with anti-HA antibodies indicated that all the SPO20 forms were present in comparable amounts (unpublished data). Therefore, the results reflect differences in the ability of the different forms to promote vesicle fusion, not differences in protein stability.

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