Althoff S M, Stevens S W, Wise J A
Department of Biochemistry, University of Illinois, Urbana 61801.
Mol Cell Biol. 1994 Dec;14(12):7839-54. doi: 10.1128/mcb.14.12.7839-7854.1994.
Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane. Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP. One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S. pombe. Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S. pombe SRP functions in protein targeting. In common with other Srp54 homologs, the S. pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain. We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually. Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein. Two mutations (R to L at position 194 [R194L] and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C. Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins. In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein. These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.
信号识别颗粒（SRP）是一种细胞质核糖核蛋白，它是将一部分分泌前体蛋白靶向内质网（ER）膜所必需的。在此，我们报告了一系列实验的结果，这些实验旨在确定裂殖酵母中哺乳动物SRP 54 kDa亚基同源物的功能。一步基因敲除实验表明，与SRP RNA一样，Srp54蛋白对于裂殖酵母的生存能力至关重要。在受调控启动子控制下，分泌蛋白酸性磷酸酶的前体在Srp54合成被抑制的细胞中积累，这表明裂殖酵母SRP在蛋白质靶向中发挥作用。与其他Srp54同源物一样，裂殖酵母蛋白具有模块化结构，由一个氨基末端G（GTP酶）结构域和一个羧基末端M（富含甲硫氨酸）结构域组成。我们分析了17个位点特异性突变的影响，这些突变旨在分别改变四个GTP酶共有基序中每一个的功能。几个等位基因，包括一些具有相对保守氨基酸替代的等位基因，赋予致死或条件性表型，这表明GTP结合和水解对于该蛋白在体内的作用至关重要。通过类比大鼠中的致癌突变设计的两个突变（第194位的R突变为L [R194L] 和R194H），旨在显著降低催化速率，还有一个突变（T248N）预计会改变核苷酸结合特异性，这些突变产生的蛋白在18℃时无法支持生长。与其设计一致，在对免疫沉淀蛋白进行的酶促测定中，R194L突变体相对于野生型Srp54以较低的速率水解GTP。在同时含有野生型srp54的菌株中，这种突变蛋白以及其他设计为锁定在GTP结合构象的蛋白，对生长表现出温度依赖性的显性抑制作用，而预计为GDP锁定的突变体则不干扰野生型蛋白的功能。这些结果构成了一个关于Srp54在SRP循环中GTP水解作用的简单模型的基础。
