Phosphate-binding loop and Rab GTPase function:: mutations at Ser29 and Ala30 of Rab5 lead to loss-of-function as well as gain-of-function phenotype
Phosphate-binding loop and Rab GTPase function:: mutations at Ser29 and Ala30 of Rab5 lead to loss-of-function as well as gain-of-function phenotype
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DOI：
10.1042/bj3550681
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发表时间：
2001-05-01
影响因子：
4.1
通讯作者：
Liang, ZM
中科院分区：
文献类型：
--
作者：
Li, GP;Liang, ZM
RAS类GTP酶含有一个结构保守的GTP结合域。GTP结合域的一个重要元件是磷酸结合环，它在RAS中含有两个Gly残基(Gly(12)和Gly(13))。由于这两个甘氨酸残基对正常的RAS功能至关重要，有趣的是，它们在其他类似RAS的GTP酶中不是保守的，包括Rab GTP酶；例如，Rab5中的等量残基是Ser(29)和Ala(30)。本研究建立在早期含有Ala(30)处取代的Rab5突变体的生化特征的基础上，并对Rab5磷酸结合环的结构与功能关系进行了全面的分析。我们已经产生了19个新的含有Ser29位氨基酸替换的突变体，并确定了这些Ser(29)突变体以及Ala(30)突变体是否仍然能够刺激幼年仓鼠肾脏细胞中的内吞精灵辣根过氧化物酶。共有11个突变体失去了刺激内吞作用的活性。在这11个突变体中，有9个是膜结合缺陷。相比之下，27个突变体仍然能够刺激内吞作用。其中五种诱导了一种新的细胞表型：细胞变圆并从培养皿中脱落。它们还可以诱导超大的早期内体，如结构性激活的Rab5：Q79L突变体。生化结果表明，Rab5的结构性激活需要增加核苷酸交换率和/或降低GTPase活性。本研究确立了Rab5磷酸结合环的功能意义，并表明该区域的突变会导致功能丧失或功能获得表型，表明一种不同于RAS的结构-功能关系。
Ras-like GTPases contain a structurally conserved GTP-binding domain. An important element of the GTP-binding domain is the phosphate-binding loop, which contains two Gly residues (Gly(12) and Gly(13)) in Ras. Because the two Gly residues are crucial for normal Ras function, it is intriguing that they are not conserved in other Ras-like GTPases, including the Rab GTPases; for example, the equivalent residues in Rab5 are Ser(29) and Ala(30). The present study builds On earlier biochemical characterizations of the Rab5 mutants containing substitutions at Ala(30) and provides a comprehensive analysis of the structure-function relationship of the Rab5 phosphate-binding loop. We have generated 19 new mutants containing amino acid substitutions at Ser29 and determined whether these Ser(29) mutants, as well as the Ala(30) mutants, remain able to stimulate the endocytosis elf horseradish peroxidase in baby hamster kidney cells. A total of 11 mutants lose the activity of stimulating endocytosis. Of these 11 mutants, 9 are defective in membrane association. In contrast, 27 mutants remain able to stimulate endocytosis. Five of them induce a novel cellular phenotype: cell rounding and detachment from culture dishes. They also induce super-large early endosomes such as the constitutively activated Rab5:Q79L mutant. Biochemical results suggest that the constitutive activation of Rab5 requires an increased nucleotide exchange rate and/or decreased GTPase activity. This study establishes functional significance for the phosphate-binding loop of Rab5 and shows that mutations in this region lead to either a loss-of-function or a gain-of-function phenotype, indicating a structure-function relationship distinct from that of Ras.