DeHoratius C, Silver P A
Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
Mol Biol Cell. 1996 Nov;7(11):1835-55. doi: 10.1091/mbc.7.11.1835.
To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport.
为了鉴定参与核蛋白输入的成分，我们采用遗传筛选方法来分离使核靶向蛋白定位错误的突变体。我们鉴定出了温度敏感型突变体，当转移至30℃的半允许温度时，这些突变体会在细胞质中积累几种不同的核蛋白；这些突变体被称为npl（核蛋白定位）突变体。我们现在展示了携带NPL4基因突变的酵母菌株的特性，并报告了NPL4基因的克隆以及Np14蛋白的特征。npl4-1突变体是通过先前描述的筛选方案分离得到的。第二个等位基因npl4-2是从独立获得的温度敏感型突变体库中鉴定出来的。npl4-1和npl4-2菌株在非允许温度下会在细胞质中积累核靶向蛋白，这与核蛋白输入缺陷一致。通过体外核输入试验，我们发现从温度转换后的npl4突变体细胞制备的细胞核即使在存在野生型细胞制备的细胞质溶胶的情况下也无法输入核靶向蛋白。此外，npl4-2细胞在非允许温度下会在细胞核中积累多聚腺苷酸（poly(A)+）RNA，这与无法将mRNA从细胞核输出一致。npl4-1和npl4-2细胞还表现出明显的、温度敏感型的结构缺陷：npl4-1细胞会向细胞质中突出额外核膜，而npl4-2细胞会形成似乎充满了poly(A)+ RNA的核膜疝。NPL4基因编码一种必需的64,000分子量的蛋白质，该蛋白质位于核周边，其定位模式与核孔复合体蛋白相似。综上所述，这些结果表明该基因编码一种新型核孔复合体或与核孔复合体相关的成分，是核膜完整性和核运输所必需的。
