Eisinger D P, Dick F A, Denke E, Trumpower B L
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Mol Cell Biol. 1997 Sep;17(9):5146-55. doi: 10.1128/MCB.17.9.5146.
QSR1 is an essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein required for joining of 40S and 60S subunits. Truncations of QSR1 predicted to encode C-terminally truncated forms of Qsr1p do not substitute for QSR1 but do act as dominant negative mutations, inhibiting the growth of yeast when expressed from an inducible promoter. The dominant negative mutants exhibit a polysome profile characterized by 'half-mer' polysomes, indicative of a subunit joining defect like that seen in other qsr1 mutants (D. P. Eisinger, F. A. Dick, and B. L. Trumpower, Mol. Cell. Biol. 17:5136-5145, 1997.) By screening a high-copy yeast genomic library, we isolated several clones containing overlapping inserts of a novel gene that rescues the slow-growth phenotype of the dominant negative qsr1 truncations. The suppressor of qsr1 truncation mutants, SQT1, is an essential gene, which encodes a 47.1-kDa protein containing multiple WD repeats and which interacts strongly with Qsr1p in a yeast two-hybrid system. SQT1 restores growth and the "half-mer" polysome profile of the dominant negative qsr1 mutants to normal, but it does not rescue temperature-sensitive qsr1 mutants or the original qsr1-1 missense allele. In yeast cell lysates, Sqt1p fractionates as part of an oligomeric protein complex that is loosely associated with ribosomes but is distinct from known eukaryotic initiation factor complexes. Loss of SQT1 function by down regulation from an inducible promoter results in formation of half-mer polyribosomes and decreased Qsr1p levels on free 60S subunits. Sqt1p thus appears to be involved in a late step of 60S subunit assembly or modification in the cytoplasm.
QSR1是酿酒酵母中的一个必需基因,它编码一种60S核糖体亚基蛋白,该蛋白是40S和60S亚基结合所必需的。预测编码Qsr1p C末端截短形式的QSR1截短体不能替代QSR1,但可作为显性负突变起作用,当从诱导型启动子表达时会抑制酵母生长。显性负突变体表现出以“半聚体”多核糖体为特征的多核糖体图谱,这表明存在类似于其他qsr1突变体中所见的亚基结合缺陷(D.P.艾辛格、F.A.迪克和B.L.特朗普沃尔,《分子细胞生物学》17:5136 - 5145,1997年)。通过筛选高拷贝酵母基因组文库,我们分离出了几个克隆,这些克隆包含一个新基因的重叠插入片段,该基因可挽救显性负性qsr1截短体的生长缓慢表型。qsr1截短突变体的抑制因子SQT1是一个必需基因,它编码一种含有多个WD重复序列的47.1 kDa蛋白,并且在酵母双杂交系统中与Qsr1p强烈相互作用。SQT1将显性负性qsr1突变体的生长和“半聚体”多核糖体图谱恢复正常,但不能挽救温度敏感型qsr1突变体或原始的qsr1 - 1错义等位基因。在酵母细胞裂解物中,Sqt1p作为寡聚蛋白复合物的一部分进行分级分离,该复合物与核糖体松散结合,但与已知的真核起始因子复合物不同。通过从诱导型启动子下调来丧失SQT1功能会导致半聚体多核糖体的形成以及游离60S亚基上Qsr1p水平的降低。因此,Sqt1p似乎参与了细胞质中60S亚基组装或修饰的后期步骤。