Santos C, Ballesta J P
Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientícas, Canto Blanco, Madrid, Spain.
J Biol Chem. 1994 Jun 3;269(22):15689-96.
Protein P0 in Saccharomyces cerevisiae is found only in the ribosomes and not free in a cytoplasmic pool like the structurally related acidic P1 and P2 proteins. Analogously, P0 stays bound to the particles in conditions that release the other P proteins. Attempts to obtain yeast strains carrying an interrupted P0 gene by direct gene disruption techniques of different yeast strains always resulted in haploid cells carrying one disrupted and one intact copy of the gene. Disruption of the unique P0 genomic copy seems to induce a duplication and occasionally a chromosomal transposition of the gene. Conditional null mutants of P0 were then constructed carrying the P0 gene under the control of the inducible GAL1 promoter. A 2-3-fold excess of P0 mRNA is found in the conditional mutant when grown in galactose; however, only a small increase of the P0 protein is detected in total cell extracts. No P0 protein is detected in the cell supernatant, indicating that, like the standard ribosomal proteins and opposite to the other P proteins, the protein not bound to the ribosomes is degraded. Transfer of the mutants to the restrictive conditions causes, after some generations, a growth stop that finally leads to cell death. The growth decline is paralleled by a reduction in the polysome number and the appearance of half-mer particles as well as by an accumulation of 60 S particles deficient in P0 and in the acidic proteins P1 and P2. These results indicate that P0 is required for the interaction of the acidic P1 and P2 proteins with the ribosomes, and in its absence, deficient 60 S ribosomes are assembled which are inactive in protein synthesis resulting in cell lethality.
酿酒酵母中的蛋白质P0仅存在于核糖体中,不像结构相关的酸性P1和P2蛋白那样游离于细胞质池中。类似地,在能释放其他P蛋白的条件下,P0仍与颗粒结合。通过不同酵母菌株的直接基因破坏技术试图获得携带中断P0基因的酵母菌株,结果总是得到携带该基因一个破坏拷贝和一个完整拷贝的单倍体细胞。独特的P0基因组拷贝的破坏似乎会诱导该基因的复制,偶尔还会导致染色体转座。然后构建了P0的条件性缺失突变体,其P0基因受可诱导的GAL1启动子控制。当在半乳糖中生长时,在条件性突变体中发现P0 mRNA过量2 - 3倍;然而,在总细胞提取物中仅检测到P0蛋白有少量增加。在细胞上清液中未检测到P0蛋白,这表明,与标准核糖体蛋白一样且与其他P蛋白相反,未与核糖体结合的蛋白会被降解。将突变体转移到限制条件下,经过几代后会导致生长停止,最终导致细胞死亡。生长下降伴随着多核糖体数量的减少和半聚体颗粒的出现,以及缺乏P0以及酸性蛋白P1和P2的60 S颗粒的积累。这些结果表明,P0是酸性P1和P2蛋白与核糖体相互作用所必需的,在其缺失时,会组装出缺乏活性的60 S核糖体,从而导致细胞死亡。