Dfg5在酿酒酵母细胞壁合成中的功能分析

Characterization of DFG5,Involved in Cell Wall Biogenesis of Saccharomyces cerevisiae

酿酒酵母中合成的糖基磷脂酰肌醇锚定蛋白(GPI-APs)一半停留在细胞膜上,另一半被转运至细胞壁的β-1,6-葡聚糖上形成GPI锚定细胞壁蛋白(CWPs)。有分析指出,Dfg5和Dcw1是定位于细胞膜的GPI锚定蛋白,可能以同工酶的形式参与GPI-APs转运。但这两个同源蛋白的分子作用机制尚不清楚。为探究Dfg5的作用机制,本研究构建了dfg5?驻PMET3DCW1条件突变菌株。研究发现在DCW1表达抑制型突变株中Cwp1在细胞膜上大量积累。对Dfg5中与甘露糖苷酶催化活性相关保守氨基酸位点分别定点突变,产生的突变蛋白Dfg5W71A、Dfg5D122A、Dfg5D123A无法回补条件突变株的生长缺陷。然而截断Dfg5前导肽的分泌型Dfg5则可回补条件突变株的生长缺陷。上述结果可推测在GPI-APs锚定至细胞壁中,Dfg5至少具有糖苷酶,可将Cwps从细胞膜上的GPI锚中释放出来。

Saccharomyces cerevisiae possesses cell wall-localized proteins(CWPs),which are essential for the growth.The majority of CWPs are initially synthesized as glycosylphosphatidylinositol(GPI)-anchored proteins,followed by transfer of the protein part to theβ-glucan.Two GPI-anchored proteins localized at the plasma membrane,Dfg5 and Dcw1,have been proposed to participate in the transfer reaction,however,molecular functions of these homologous proteins are still unclear.In this study,we characterized the Dfg5 using a conditional mutant strain in which DFG5 was disrupted and DCW1 expression was controlled under MET3 promoter.We found that down-regulation of DCW1 expression in the mutant strain caused a great accumulation of our model CWP(Cwp1)in the plasma membrane.Site-directed mutagenesis of Dfg5 revealed that three residues conserved in glycoside hydrolase family 76 were necessary to complement the lethality of the mutant strain.In contrast,the growth defect was complemented by the expression of soluble form of Dfg5,which was secreted in culture medium due to a lack of its pro-peptide sequence.Taken together,these results suggested that at least Dfg5 acts as an enzyme to release CWPs from GPI anchor at plasma membrane.