G(1) CYCLIN DEGRADATION - THE PEST MOTIF OF YEAST CLN2 IS NECESSARY, BUT NOT SUFFICIENT, FOR RAPID PROTEIN-TURNOVER
G(1) CYCLIN DEGRADATION - THE PEST MOTIF OF YEAST CLN2 IS NECESSARY, BUT NOT SUFFICIENT, FOR RAPID PROTEIN-TURNOVER
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DOI：
10.1128/mcb.14.12.7953
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发表时间：
1994-12-01
影响因子：
5.3
通讯作者：
THORNER, J
中科院分区：
文献类型：
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作者：
SALAMA, SR;HENDRICKS, KB;THORNER, J
545个残基的Cln 2蛋白，像酿酒酵母的其他G（1）细胞周期蛋白一样，是一种非常不稳定的蛋白。这种不稳定性被认为在调节细胞周期进程中起关键作用。Cln 2和其他G（1）细胞周期蛋白的羧基末端结构域含有富含Pro、Glu（和Asp）、Ser和Thr的序列（所谓的PEST基序），这些序列被认为是构成这些和其他不稳定蛋白快速降解的信号的原因。为了检验这一假设，将Cln 2的羧基末端178个残基融合到报告酶的C末端，所述报告酶是人胸苷激酶（hTK Delta 40）的截短形式。所得的嵌合蛋白（hTK Delta 40-Cln 2）保留胸苷激酶活性，但明显不如hTK，hTK Delta 40，或hTK-β-半乳糖苷酶融合蛋白稳定，如通过酶测定，抗hTK抗体免疫印迹，放射性标记多肽的脉冲追踪分析，以及支持胸苷酸营养缺陷型（cdc 21突变体）在含胸苷培养基上生长的能力。因此，Cln 2 PEST结构域的存在足以使异源蛋白不稳定。此外，hTK Delta 40-Cln 2的半衰期与真实Cln 2的半衰期相似，并且已知引起G（1）停滞的处理（包括将MATa单倍体暴露于α因子交配信息素和将cdc 28（ts）和cdc 34（ts）突变体转移至限制性温度）均未显著提高蛋白质的降解速率。这些结果表明，负责Cln 2不稳定性的主要信号仅限于其C-末端三分之一。因为hTK Delta 40-Cln 2和Cln 2由异源启动子表达，但它们在异步培养物中的半衰期和当在各种细胞周期阶段停滞时的半衰期总是相似的，所以Cln 2，PEST结构域含有快速蛋白质周转的信号，其在整个细胞周期中是组成型活性和有效的。从hTK Delta 40-Cln 2或Cln 2中去除编码最突出的PEST样片段的37个密码子降低了所得蛋白质的周转率，正如预期的那样;然而，仅含有该37个残基片段的hTK Delta 40嵌合体没有可检测的不稳定性，表明该PEST序列在从其正常环境中去除时不是蛋白质不稳定性的自含决定因素。
The 545-residue Cln2 protein, like the other G(1) cyclins of Saccharomyces cerevisiae, is a very unstable protein. This instability is thought to play a critical role in regulating cell cycle progression. The carboxyl-terminal domains of Cln2 and the other G(1) cyclins contain sequences rich in Pro, Glu (and Asp), Ser, and Thr (so-called PEST motifs) that have been postulated to make up the signals that are responsible for the rapid degradation of these and other unstable proteins. To test this hypothesis, the carboxyl-terminal 178 residues of Cln2 were fused to the C terminus of a reporter enzyme, a truncated form of human thymidine kinase (hTK Delta 40). The resulting chimeric protein (hTK Delta 40-Cln2) retained thymidine kinase activity but was markedly less stable than hTK, hTK Delta 40, or an hTK-beta-galactosidase fusion protein, as judged by enzyme assay, immunoblotting with anti-hTK antibodies, pulse-chase analysis of the radiolabeled polypeptides, and ability to support the growth of a thymidylate auxotroph (cdc21 mutant) on thymidine-containing medium. Thus, the presence of the Cln2 PEST domain was sufficient to destabilize a heterologous protein. Furthermore, the half-life of hTK Delta 40-Cln2 was similar to that of authentic Cln2, and the rate of degradation of neither protein was detectably enhanced by treatments known to cause G(1) arrest, including exposure of MATa haploids to alpha-factor mating pheromone and shifting cdc28(ts) and cdc34(ts) mutants to the restrictive temperature. These results suggest that the major signals responsible for Cln2 instability are confined to its C-terminal third. Because hTK Delta 40-Cln2 and Cln2 were expressed from heterologous promoters yet their half-lives both in asynchronous cultures and when arrested at various cell cycle stages were always similar, the Cln2, PEST domain contains a signal for rapid protein turnover that is constitutively active and operative throughout the cell cycle. Removal of the 37 codons that encode the most prominent PEST-like segment from either hTK Delta 40-Cln2 or Cln2 decreased the turnover rate of the resulting proteins, as expected; however, an hTK Delta 40 chimera containing only this 37-residue segment was not detectably destabilized, suggesting that this PEST sequence, when removed from its normal context, is not a self-contained determinant of protein instability.