Lee D H, Sherman M Y, Goldberg A L
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Cell Biol. 1996 Sep;16(9):4773-81. doi: 10.1128/MCB.16.9.4773.
In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.
在大肠杆菌和线粒体中，分子伴侣DnaJ不仅参与蛋白质折叠，还参与某些异常多肽的选择性降解。我们在此证明，在酵母胞质溶胶中，同源伴侣蛋白Ydj1对于某些异常蛋白质的泛素依赖性降解也是必需的。温度敏感型ydj1-151突变体在短命细胞蛋白和含氨基酸类似物的异常多肽的整体降解中表现出较大缺陷，尤其是在38摄氏度时。相比之下，与泛素无关的长寿细胞蛋白的降解未发生改变，细胞生长也未受影响。Ydj1的失活显著降低了某些β-半乳糖苷酶（β-gal）融合多肽的快速、泛素依赖性降解。尽管N端规则底物（精氨酸-β-gal和亮氨酸-β-gal）以及B型细胞周期蛋白Clb5-β-gal的降解正常进行，但异常多肽泛素-脯氨酸-β-gal（Ub-P-β-gal）和短命正常蛋白Gcn4的降解受到抑制。由于Ub-P-β-gal降解减少，温度敏感型ydj1-151突变体细胞中的β-gal活性比野生型细胞高四到五倍；因此，该酶的折叠和组装不需要Ydj1的功能。在野生型细胞中，而非ydj1-151突变体细胞中，这种伴侣蛋白与短命底物Ub-P-β-gal相关，而不与稳定的β-gal构建体相关。此外，在ydj1-151突变体中，Ub-P-β-gal的泛素化被阻断，细胞中泛素化蛋白的总水平降低。因此，Ydj1对于某些蛋白质的泛素依赖性降解至关重要。这种伴侣蛋白可能有助于识别未折叠的蛋白质，或作为某些泛素连接酶的辅助因子。
