Richardson C J, Jones S, Litt R J, Segev N
Department of Biochemistry and Molecular Biology, The University of Chicago, Illinois 60637, USA.
Mol Cell Biol. 1998 Feb;18(2):827-38. doi: 10.1128/MCB.18.2.827.
GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37 degrees C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.
Ypt/Rab家族的GTP酶在囊泡运输的调控中起关键作用。它们在结合GTP和结合GDP的形式之间循环的能力被认为对其功能至关重要。从结合GTP的形式转变为结合GDP的形式是通过微弱的内源性GTP酶活性实现的，这种活性可被GTP酶激活蛋白（GAP）刺激。目前的模型表明，GTP水解和GAP活性对于囊泡与受体区室的融合或膜融合的定时至关重要。为了验证这一观点，我们通过使用Q67L突变使Ypt1p的GTP酶活性失活，该突变靶向一个保守残基，该残基有助于催化Ras中的GTP水解。我们证明，突变的Ypt1-Q67L蛋白在不存在和存在GAP的情况下水解GTP的能力都严重受损，因此主要局限于结合GTP的形式。令人惊讶的是，一个以ypt1-Q67L作为细胞中唯一YPT1基因的菌株在14至37摄氏度的温度范围内没有可观察到的生长表型。此外，通过电子显微镜测定，这些突变细胞表现出正常的分泌速率和正常的膜形态。此外，ypt1-Q67L等位基因在过表达时在细胞生长和分泌中不表现出显性表型。总之，这些结果使我们提出，与目前关于Ypt/Rab功能的模型相反，GTP水解对于Ypt1p介导的囊泡运输或作为关闭Ypt1p介导的膜融合的定时器不是必需的，而仅对于Ypt1p在区室之间的循环是必需的。最后，ypt1-Q67L等位基因与野生型一样，受到显性无核苷酸YPT1突变的抑制。这种突变被认为通过隔离鸟嘌呤核苷酸交换因子（GNEF）发挥其显性表型。这些结果表明，Ypt1p在囊泡运输中的功能不仅需要该蛋白的结合GTP的形式，还需要Ypt1p与其GNEF的相互作用。
