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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 is a more efficient fusogen in vitro. (A) Liposome fusion assay. Liposomes containing Sso1 and the SNARE domains of Sec9, Spo20, or Spo20C224L,S231N were mixed with Snc2-containing liposomes and fusion assayed by fluorescence. (B) Stability of Sso1/Snc2/ Spo20C224L,S231N complexes in increasing concentrations of Guanidinium-HCl.
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fig8: Spo20C224L,S231N is a more efficient fusogen in vitro. (A) Liposome fusion assay. Liposomes containing Sso1 and the SNARE domains of Sec9, Spo20, or Spo20C224L,S231N were mixed with Snc2-containing liposomes and fusion assayed by fluorescence. (B) Stability of Sso1/Snc2/ Spo20C224L,S231N complexes in increasing concentrations of Guanidinium-HCl.

Mentions: Studies of Sec9- and Spo20-containing SNARE complexes in an in vitro liposome fusion system indicate that, in a given lipid composition, Spo20-containing complexes are less fusogenic than Sec9 complexes (Liu et al., 2007). Moreover, this lesser activity correlates with decreased SNARE complex stability (measured as a lower melting temperature) of the Spo20 complexes compared with Sec9 complexes. The behavior of the SPO20C224L,S231N mutant in the genetic tests and immunoprecipitations described above suggests that these mutations might increase the binding energy of the Spo20-containing complexes. To test this directly, the recombinant SNARE domain (amino acids 147–397) of Spo20C224L,S231N was purified from Escherichia coli and tested with the Sso1 and Snc2 proteins in a liposome fusion assay. Using liposomes containing 85% POPC (palmitoyl oleoyl phosphatidylcholine) and 15% DOPS (di-oleoyl phosphatidylserine), Sso1/Sec9–Snc2 complexes promote liposome fusion at a greater rate than the Sso1/Spo20–Snc2 SNAREs (Fig. 8 A). In contrast, Sso1/Spo20C224L,S231N–Snc2 mediates fusion at a rate comparable to the Sec9 complexes. Thus, parallel to the in vivo results, Spo20C224L,S231N promotes fusion more efficiently than Spo20 in vitro.


Binding interactions control SNARE specificity in vivo.

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

Spo20C224L,S231N is a more efficient fusogen in vitro. (A) Liposome fusion assay. Liposomes containing Sso1 and the SNARE domains of Sec9, Spo20, or Spo20C224L,S231N were mixed with Snc2-containing liposomes and fusion assayed by fluorescence. (B) Stability of Sso1/Snc2/ Spo20C224L,S231N complexes in increasing concentrations of Guanidinium-HCl.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Spo20C224L,S231N is a more efficient fusogen in vitro. (A) Liposome fusion assay. Liposomes containing Sso1 and the SNARE domains of Sec9, Spo20, or Spo20C224L,S231N were mixed with Snc2-containing liposomes and fusion assayed by fluorescence. (B) Stability of Sso1/Snc2/ Spo20C224L,S231N complexes in increasing concentrations of Guanidinium-HCl.
Mentions: Studies of Sec9- and Spo20-containing SNARE complexes in an in vitro liposome fusion system indicate that, in a given lipid composition, Spo20-containing complexes are less fusogenic than Sec9 complexes (Liu et al., 2007). Moreover, this lesser activity correlates with decreased SNARE complex stability (measured as a lower melting temperature) of the Spo20 complexes compared with Sec9 complexes. The behavior of the SPO20C224L,S231N mutant in the genetic tests and immunoprecipitations described above suggests that these mutations might increase the binding energy of the Spo20-containing complexes. To test this directly, the recombinant SNARE domain (amino acids 147–397) of Spo20C224L,S231N was purified from Escherichia coli and tested with the Sso1 and Snc2 proteins in a liposome fusion assay. Using liposomes containing 85% POPC (palmitoyl oleoyl phosphatidylcholine) and 15% DOPS (di-oleoyl phosphatidylserine), Sso1/Sec9–Snc2 complexes promote liposome fusion at a greater rate than the Sso1/Spo20–Snc2 SNAREs (Fig. 8 A). In contrast, Sso1/Spo20C224L,S231N–Snc2 mediates fusion at a rate comparable to the Sec9 complexes. Thus, parallel to the in vivo results, Spo20C224L,S231N promotes fusion more efficiently than Spo20 in vitro.

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