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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 the Spo20 helices increases binding of Sso1 and Snc2. Strain HI75 (sso1Δ sso2Δ) was transformed with high copy plasmids expressing 3xHA tagged forms of Δ3-51Spo20, Δ3-51Spo20C224L,S231N, or Δ3-51PSPS chimera. Additionally, these cells carried a CEN plasmid expressing either SSO1 or SSO1Q224R and high copy plasmids expressing SNC2 or SNC2R52Q, respectively. These strains were grown to mid-log in selective medium, lysed, and the HA-tagged Spo20 proteins immunoprecipitated. (A) (top) Western blots of cell lysates from each strain probed with anti-HA, anti-Sso1, or anti-Snc2 antibodies. 3X-HA indicates bands corresponding to the different Spo20 mutants; (bottom) Western blots of anti-HA immunoprecipitates from the same lysates. The band corresponding to Δ3-51PSPS in the Sso1Q224R/SncR52Q strain is shown from a longer exposure of the same blot. Asterisk indicates Sso1Q224R, which displays slightly increased mobility compared with the wild-type Sso1. (B) Quantitation of the coprecipitation of Sso1 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Sso1 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Sso1 blots. Values shown are the average of three experiments. Bars indicate one standard deviation. (C) Quantitation of the coprecipitation of Snc2 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Snc2 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Snc2 blots. Values shown are the average of three experiments. Bars indicate one standard deviation.
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fig7: Mutation of the Spo20 helices increases binding of Sso1 and Snc2. Strain HI75 (sso1Δ sso2Δ) was transformed with high copy plasmids expressing 3xHA tagged forms of Δ3-51Spo20, Δ3-51Spo20C224L,S231N, or Δ3-51PSPS chimera. Additionally, these cells carried a CEN plasmid expressing either SSO1 or SSO1Q224R and high copy plasmids expressing SNC2 or SNC2R52Q, respectively. These strains were grown to mid-log in selective medium, lysed, and the HA-tagged Spo20 proteins immunoprecipitated. (A) (top) Western blots of cell lysates from each strain probed with anti-HA, anti-Sso1, or anti-Snc2 antibodies. 3X-HA indicates bands corresponding to the different Spo20 mutants; (bottom) Western blots of anti-HA immunoprecipitates from the same lysates. The band corresponding to Δ3-51PSPS in the Sso1Q224R/SncR52Q strain is shown from a longer exposure of the same blot. Asterisk indicates Sso1Q224R, which displays slightly increased mobility compared with the wild-type Sso1. (B) Quantitation of the coprecipitation of Sso1 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Sso1 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Sso1 blots. Values shown are the average of three experiments. Bars indicate one standard deviation. (C) Quantitation of the coprecipitation of Snc2 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Snc2 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Snc2 blots. Values shown are the average of three experiments. Bars indicate one standard deviation.

Mentions: If alteration of the SNARE helices of Spo20 increases its affinity for its partner SNAREs, this should be reflected in increased binding of the protein to Sso1 and Snc2. To address this possibility, HA-tagged Δ50-Spo20, Spo20C224L,S231N, or PSPS were expressed in combination with either wild-type Sso1 and Snc2 or Sso1Q224R and Snc2R52Q in an sso1 sso2 strain. Lysates were made from each strain and the Spo20 proteins immunoprecipitated using anti-HA antibodies. Immunoprecipitates were then blotted and probed with anti-HA, anti-Sso1, or anti-Snc2 antibodies to examine association of the three SNARE proteins (Fig. 7). In the presence of both the wild-type and mutant Sso1 and Snc2 proteins the same pattern was seen; the PSPS chimera precipitated significantly more Sso1 and Snc2 than Spo20C224L,S231N, which in turn brought down slightly more Sso1 and Snc2 than the wild-type Spo20. Though these immunoprecipitations do not provide a direct measure of affinity, the increased association of PSPS and Spo20C224L,S231N with both forms of Sso1 and Snc2 are consistent with the idea that they bind more avidly to their partner SNAREs than wild-type Spo20. Interestingly, all three forms of Spo20 exhibited greater association with the Sso1Q224R and Snc2R52Q proteins than with the wild-type SNAREs. Because the amount of SNAREs in complex reflects both the rates of assembly and of disassembly, we suggest that the general increase in the amount of SNARE complexes seen with Sso1Q224R/Snc2R52Q might reflect a role for the central ionic layer in complex disassembly, as suggested previously (Scales et al., 2001).


Binding interactions control SNARE specificity in vivo.

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

Mutation of the Spo20 helices increases binding of Sso1 and Snc2. Strain HI75 (sso1Δ sso2Δ) was transformed with high copy plasmids expressing 3xHA tagged forms of Δ3-51Spo20, Δ3-51Spo20C224L,S231N, or Δ3-51PSPS chimera. Additionally, these cells carried a CEN plasmid expressing either SSO1 or SSO1Q224R and high copy plasmids expressing SNC2 or SNC2R52Q, respectively. These strains were grown to mid-log in selective medium, lysed, and the HA-tagged Spo20 proteins immunoprecipitated. (A) (top) Western blots of cell lysates from each strain probed with anti-HA, anti-Sso1, or anti-Snc2 antibodies. 3X-HA indicates bands corresponding to the different Spo20 mutants; (bottom) Western blots of anti-HA immunoprecipitates from the same lysates. The band corresponding to Δ3-51PSPS in the Sso1Q224R/SncR52Q strain is shown from a longer exposure of the same blot. Asterisk indicates Sso1Q224R, which displays slightly increased mobility compared with the wild-type Sso1. (B) Quantitation of the coprecipitation of Sso1 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Sso1 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Sso1 blots. Values shown are the average of three experiments. Bars indicate one standard deviation. (C) Quantitation of the coprecipitation of Snc2 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Snc2 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Snc2 blots. Values shown are the average of three experiments. Bars indicate one standard deviation.
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fig7: Mutation of the Spo20 helices increases binding of Sso1 and Snc2. Strain HI75 (sso1Δ sso2Δ) was transformed with high copy plasmids expressing 3xHA tagged forms of Δ3-51Spo20, Δ3-51Spo20C224L,S231N, or Δ3-51PSPS chimera. Additionally, these cells carried a CEN plasmid expressing either SSO1 or SSO1Q224R and high copy plasmids expressing SNC2 or SNC2R52Q, respectively. These strains were grown to mid-log in selective medium, lysed, and the HA-tagged Spo20 proteins immunoprecipitated. (A) (top) Western blots of cell lysates from each strain probed with anti-HA, anti-Sso1, or anti-Snc2 antibodies. 3X-HA indicates bands corresponding to the different Spo20 mutants; (bottom) Western blots of anti-HA immunoprecipitates from the same lysates. The band corresponding to Δ3-51PSPS in the Sso1Q224R/SncR52Q strain is shown from a longer exposure of the same blot. Asterisk indicates Sso1Q224R, which displays slightly increased mobility compared with the wild-type Sso1. (B) Quantitation of the coprecipitation of Sso1 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Sso1 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Sso1 blots. Values shown are the average of three experiments. Bars indicate one standard deviation. (C) Quantitation of the coprecipitation of Snc2 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Snc2 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Snc2 blots. Values shown are the average of three experiments. Bars indicate one standard deviation.
Mentions: If alteration of the SNARE helices of Spo20 increases its affinity for its partner SNAREs, this should be reflected in increased binding of the protein to Sso1 and Snc2. To address this possibility, HA-tagged Δ50-Spo20, Spo20C224L,S231N, or PSPS were expressed in combination with either wild-type Sso1 and Snc2 or Sso1Q224R and Snc2R52Q in an sso1 sso2 strain. Lysates were made from each strain and the Spo20 proteins immunoprecipitated using anti-HA antibodies. Immunoprecipitates were then blotted and probed with anti-HA, anti-Sso1, or anti-Snc2 antibodies to examine association of the three SNARE proteins (Fig. 7). In the presence of both the wild-type and mutant Sso1 and Snc2 proteins the same pattern was seen; the PSPS chimera precipitated significantly more Sso1 and Snc2 than Spo20C224L,S231N, which in turn brought down slightly more Sso1 and Snc2 than the wild-type Spo20. Though these immunoprecipitations do not provide a direct measure of affinity, the increased association of PSPS and Spo20C224L,S231N with both forms of Sso1 and Snc2 are consistent with the idea that they bind more avidly to their partner SNAREs than wild-type Spo20. Interestingly, all three forms of Spo20 exhibited greater association with the Sso1Q224R and Snc2R52Q proteins than with the wild-type SNAREs. Because the amount of SNAREs in complex reflects both the rates of assembly and of disassembly, we suggest that the general increase in the amount of SNARE complexes seen with Sso1Q224R/Snc2R52Q might reflect a role for the central ionic layer in complex disassembly, as suggested previously (Scales et al., 2001).

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