Martínez-Force E, Lakhe-Reddy S, Wise J A
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4960, USA.
Curr Genet. 1999 Mar;35(2):88-102. doi: 10.1007/s002940050437.
The signal recognition particle (SRP) is a ribonucleoprotein required for targeting a subset of nascent pre-secretory proteins to the endoplasmic reticulum membrane. Of the six SRP polypeptides, the most highly conserved is Srp54p, a modular protein consisting of an amino-terminal (N) domain of unknown function, a central GTPase (G) domain, and a carboxyl-terminal (M) domain implicated in the recognition of both signal sequences and SRP RNA. To identify regions of Srp54p that interact with other SRP subunits or regulatory proteins, we carried out systematic mutagenesis of the fission yeast homolog, principally using a "clustered charged-to-alanine" strategy. Of the 35 alleles examined, 13 are unable to support growth, two confer cold-sensitivity, five confer heat-sensitivity, and 15 produce no discernible phenotype. The lethal and conditional mutations map throughout the protein to several conserved regions, confirming that these motifs play critical roles in Srp54p function. The effects of the amino-acid substitutions are analyzed with reference to the recently determined tertiary structures of the N/G domain and the intact protein from a thermophilic bacterium.
信号识别颗粒（SRP）是一种核糖核蛋白，它对于将一部分新生的前分泌蛋白靶向内质网膜是必需的。在六种SRP多肽中，保守性最高的是Srp54p，它是一种模块化蛋白，由一个功能未知的氨基末端（N）结构域、一个中央GTP酶（G）结构域和一个羧基末端（M）结构域组成，该羧基末端结构域参与信号序列和SRP RNA的识别。为了鉴定Srp54p中与其他SRP亚基或调节蛋白相互作用的区域，我们主要使用“成簇电荷到丙氨酸”策略对裂殖酵母同源物进行了系统诱变。在检测的35个等位基因中，13个不能支持生长，2个导致冷敏感性，5个导致热敏感性，15个没有产生可识别的表型。致死和条件性突变遍布整个蛋白质，定位于几个保守区域，证实这些基序在Srp54p功能中起关键作用。参考最近确定的嗜热细菌N/G结构域和完整蛋白质的三级结构，分析了氨基酸取代的影响。
