Research Program in Cell and Molecular Biology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
PLoS One. 2010 Oct 11;5(10):e13323. doi: 10.1371/journal.pone.0013323.
The Saccharomyces cerevisiae syntaxin1 homologues Sso1p and Sso2p perform an essential function in membrane fusion in exocytosis. While deletion of either SSO1 or SSO2 causes no obvious phenotype in vegetatively grown cells, deletion of both genes is lethal. In sporulating diploid S. cerevisiae cells only Sso1p, but not Sso2p, is needed for membrane fusion during prospore membrane formation. Mass spectrometry and in vivo labeling data suggest that serines 23, 24, and 79 in Sso1p and serines 31 and 34 in Sso2p can be phosphorylated in vivo. Here we set out to assess the contribution of phosphorylation on Sso protein in vivo function.
Different mutant versions of SSO1 and SSO2 were generated to target the phosphorylation sites in Sso1p and Sso2p. Basal or overexpression of phospho-mimicking or putative non-phosphorylated Sso1p or Sso2p mutants resulted in no obvious growth phenotype. However, S79A and S79E mutations caused a mild defect in the ability of Sso1p to complement the temperature-sensitive growth phenotype of sso2-1 sso1Δ cells. Combination of all mutations did not additionally compromise Sso1p in vivo function. When compared to the wild type SSO1 and SSO2, the phosphoamino acid mutants displayed similar genetic interactions with late acting sec mutants. Furthermore, diploid cells expressing only the mutant versions of Sso1p had no detectable sporulation defects. In addition to sporulation, also pseudohyphal and invasive growth modes are regulated by the availability of nutrients. In contrast to sporulating diploid cells, deletion of SSO1 or SSO2, or expression of the phospho-mutant versions of SSO1 or SSO2 as the sole copies of SSO genes caused no defects in haploid or diploid pseudohyphal and invasive growth.
The identified phosphorylation sites do not significantly contribute to the in vivo functionality of Sso1p and Sso2p in S. cerevisiae.
酿酒酵母的突触蛋白 1 同源物 Sso1p 和 Sso2p 在胞吐作用中的膜融合中发挥重要作用。虽然删除 SSO1 或 SSO2 中的任何一个都不会在营养生长的细胞中引起明显的表型，但两个基因的缺失都是致命的。在产孢子的二倍体酿酒酵母细胞中，只有 Sso1p，而不是 Sso2p，在孢子膜形成过程中需要进行膜融合。质谱分析和体内标记数据表明，Sso1p 中的丝氨酸 23、24 和 79 以及 Sso2p 中的丝氨酸 31 和 34 可以在体内发生磷酸化。在这里，我们着手评估 Sso 蛋白磷酸化对其体内功能的贡献。
生成了不同的 SSO1 和 SSO2 突变版本，以靶向 Sso1p 和 Sso2p 中的磷酸化位点。基础表达或过表达磷酸模拟或假定非磷酸化的 Sso1p 或 Sso2p 突变体不会导致明显的生长表型。然而，S79A 和 S79E 突变导致 Sso1p 补充 sso2-1 sso1Δ 细胞温度敏感生长表型的能力出现轻微缺陷。所有突变的组合并没有进一步损害 Sso1p 的体内功能。与野生型 SSO1 和 SSO2 相比，磷酸氨基酸突变体与晚期作用的 sec 突变体表现出相似的遗传相互作用。此外，与野生型 SSO1 和 SSO2 相比，只表达突变体版本 Sso1p 的二倍体细胞没有检测到明显的孢子形成缺陷。除了孢子形成外，营养物质的可用性还调节假菌丝和侵袭性生长模式。与产孢子的二倍体细胞不同，删除 SSO1 或 SSO2，或仅作为 SSO 基因的拷贝表达磷酸突变体版本的 SSO1 或 SSO2，不会导致单倍体或二倍体假菌丝和侵袭性生长缺陷。
鉴定的磷酸化位点对酿酒酵母中 Sso1p 和 Sso2p 的体内功能没有显著贡献。
