In Saccharomyces cerevisiae, diploid yeast cells follow a bipolar budding program, which depends on the two transmembrane glycoproteins Bud8p and Bud9p that potentially act as cortical tags to mark the cell poles. Here, we have performed systematic structure-function analyses of Bud8p and Bud9p to identify functional domains. We find that polar transport of Bud8p and Bud9p does not depend on N-terminal sequences but instead on sequences in the median part of the proteins and on the C-terminal parts that contain the transmembrane domains. We show that the guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange factor Bud5p, which is essential for bud site selection and physically interacts with Bud8p, also interacts with Bud9p. Regions of Bud8p and Bud9p predicted to reside in the extracellular space are likely to confer interaction with the N-terminal region of Bud5p, implicating indirect interactions between the cortical tags and the GDP/GTP exchange factor. Finally, we have identified regions of Bud8p and Bud9p that are required for interaction with the cortical tag protein Rax1p. In summary, our study suggests that Bud8p and Bud9p carry distinct domains for delivery of the proteins to the cell poles, for interaction with the general budding machinery and for association with other cortical tag proteins.

译文

在酿酒酵母中,二倍体酵母细胞遵循双极芽接程序,该程序取决于两个跨膜糖蛋白Bud8p和Bud9p,它们可能充当皮质标签来标记细胞极。在这里,我们进行了Bud8p和Bud9p的系统结构-功能分析,以识别功能域。我们发现Bud8p和Bud9p的极性转运不依赖于N末端序列,而是依赖于蛋白质中间部分的序列以及包含跨膜结构域的C末端部分。我们表明,鸟苷二磷酸(GDP)/鸟苷三磷酸(GTP)交换因子Bud5p,这对于芽位点选择至关重要,并且与Bud8p物理相互作用,也与Bud9p相互作用。预计驻留在细胞外空间中的Bud8p和Bud9p区域可能会赋予与Bud5p的N端区域相互作用,这暗示皮层标签与GDP / GTP交换因子之间存在间接相互作用。最后,我们确定了与皮质标签蛋白Rax1p相互作用所需的Bud8p和Bud9p区域。总而言之,我们的研究表明Bud8p和Bud9p携带不同的结构域,用于将蛋白质递送至细胞极,与一般的出芽机制相互作用以及与其他皮质标签蛋白结合。

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