Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae
Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae
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DOI：
10.1073/pnas.95.10.5584
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发表时间：
1998-05-12
影响因子：
11.1
通讯作者：
Linder, ME
中科院分区：
文献类型：
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作者：
Srinivasa, SP;Bernstein, LS;Linder, ME
RGS4是一种作用于G蛋白α亚基的哺乳动物GTP酶激活蛋白，它是通过其抑制酿酒酵母信息素应答途径的能力而被鉴定出来的。为了确定RGS4在体内发挥功能所必需的区域，我们检测了该系统中突变体的活性。RGS4的N末端33个氨基酸缺失（Δ1 - 33）产生了一种无功能的蛋白质，并且失去了质膜定位。通过向RGS4（Δ1 - 33）添加一个C末端膜靶向序列，这些功能得以恢复。因此，质膜定位与RGS4抑制信号传导的能力紧密相关。将RGS4的N末端33个氨基酸与绿色荧光蛋白融合，足以将一种原本可溶的蛋白质定位到质膜上，这表明该N末端区域是一个质膜锚定结构域。RGS4是棕榈酰化的，Cys - 2和Cys - 12可能是棕榈酰化位点。令人惊讶的是，RGS4的N末端结构域内半胱氨酸残基的突变并没有影响其在酵母中的质膜定位或抑制信号传导的能力。RGS4的N末端结构域中除棕榈酰化之外的特征是其与质膜结合以及在酵母中抑制信息素应答能力的原因。
RGS4, a mammalian GTPase activating protein for G protein alpha subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae. To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system. Deletion of the N-terminal 33 aa of RGS4 (Delta 1-33) yielded a nonfunctional protein and loss of plasma membrane localization. These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (Delta 1-33). Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to green fluorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain. RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation, Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did Plot affect plasma membrane localization in yeast or the ability to inhibit signaling. Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.