Dockendorff T C, Heath C V, Goldstein A L, Snay C A, Cole C N
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Mol Cell Biol. 1997 Feb;17(2):906-20. doi: 10.1128/MCB.17.2.906.
A screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective in nucleocytoplasmic trafficking of poly(A)+ RNA has identified an allele of the NUP145 gene, which encodes an essential nucleoporin. NUP145 was previously identified by using a genetic synthetic lethal screen (E. Fabre, W. C. Boelens, C. Wimmer, I. W. Mattaj, and E. C. Hurt, Cell 78:275-289, 1994) and by using a monoclonal antibody which recognizes the GLFG family of vertebrate and yeast nucleoporins (S. R. Wente and G. Blobel, J. Cell Biol. 125:955-969, 1994). Cells carrying the new allele, nup145-10, grew at 23 and 30 degrees C but were unable to grow at 37 degrees C. Many cells displayed a modest accumulation of poly(A)+ RNA under permissive growth conditions, and all cells showed dramatic and rapid nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. The mutant allele contains a nonsense codon which truncates the 1,317-amino-acid protein to 698 amino acids. This prompted us to examine the role of the carboxyl half of Nup145p. Several additional alleles that encode C-terminally truncated proteins or proteins containing internal deletions of portions of the carboxyl half of Nup145p were constructed. Analysis of these mutants indicates that some sequences between amino acids 698 and 1095 are essential for RNA export and for growth at 37 degrees C. In these strains, nuclear accumulation of poly(A)+ RNA and fragmentation of the nucleolus occurred rapidly following a shift to 37 degrees C. Constitutive defects in nuclear pore complex distribution and nuclear structure were also seen in these strains. Although cells lacking Nup145p grew extremely slowly at 23 degrees C and did not grow at 30 degrees C, efficient growth at 23 or 30 degrees C occurred as long as cells produced either the amino 58% or the carboxyl 53% of Nup145p. Strains carrying alleles of NUP145 lacking up to 200 amino acids from the carboxy terminus were viable at 37 degrees C but displayed nucleolar fragmentation and some nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. Surprisingly, these strains grew efficiently at 37 degrees C in spite of a reduction in the level of synthesis of rRNAs to approximately 25% of the wild-type level.
通过对酿酒酵母中聚腺苷酸（poly(A)+）RNA核质运输缺陷的温度敏感突变体进行筛选，鉴定出NUP145基因的一个等位基因，该基因编码一种必需的核孔蛋白。NUP145此前是通过基因合成致死筛选（E. Fabre、W. C. Boelens、C. Wimmer、I. W. Mattaj和E. C. Hurt，《细胞》78:275 - 289，1994年）以及使用识别脊椎动物和酵母核孔蛋白GLFG家族的单克隆抗体（S. R. Wente和G. Blobel，《细胞生物学杂志》125:955 - 969，1994年）鉴定出来的。携带新等位基因nup145 - 10的细胞在23℃和30℃下能够生长，但在37℃下无法生长。许多细胞在允许生长条件下聚腺苷酸（poly(A)+）RNA有适度积累，并且所有细胞在转移至37℃后聚腺苷酸（poly(A)+）RNA都迅速在细胞核中大量积累。该突变等位基因包含一个无义密码子，它将1317个氨基酸的蛋白质截短为698个氨基酸。这促使我们研究Nup145p羧基端一半的作用。构建了几个额外的等位基因，它们编码C端截短的蛋白质或包含Nup145p羧基端一半部分内部缺失的蛋白质。对这些突变体的分析表明，氨基酸698至1095之间的一些序列对于RNA输出和在37℃下生长是必需的。在这些菌株中，转移至37℃后聚腺苷酸（poly(A)+）RNA迅速在细胞核中积累且核仁发生碎片化。在这些菌株中还观察到核孔复合体分布和核结构的组成性缺陷。尽管缺乏Nup145p的细胞在23℃下生长极其缓慢且在30℃下无法生长，但只要细胞产生Nup145p的氨基端58%或羧基端53%，在23℃或30℃下就能有效生长。携带从羧基末端缺失多达200个氨基酸的NUP145等位基因的菌株在37℃下是存活的，但转移至37℃后显示核仁碎片化和聚腺苷酸（poly(A)+）RNA在细胞核中的一些积累。令人惊讶的是，尽管这些菌株中rRNA的合成水平降至野生型水平的约25%，但它们在37℃下仍能有效生长。
