Graduate School of Medical Life Science and Advanced Medical Research Center, Yokohama City University, Yokohama, Japan.
Proteomics. 2013 Nov;13(21):3167-74. doi: 10.1002/pmic.201300207. Epub 2013 Oct 9.
The 26S proteasome is a multicatalytic protease complex that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core (the 20S proteasome) as well as regulatory particles, which contain six ATPase (Rpt) subunits involved in unfolding and translocation of substrates to the catalytic chamber of the 20S proteasome. In this study, we used MS to analyze the N-terminal modifications of the yeast Rpt1 subunit, which contains the N-terminal recognition sequence for N-methyltransferase. Our results revealed that following the removal of the initiation Met residue of yeast Rpt1, the N-terminal Pro residue is either unmodified, mono-methylated, or di-methylated, and that this N-methylation has not been conserved throughout evolution. In order to gain a better understanding of the possible function(s) of the Pro-Lys (PK) sequence at positions 3 and 4 of yeast Rpt1, we generated mutant strains expressing an Rpt1 allele that lacks this sequence. The absence of the PK sequence abolished N-methylation, decreased cell growth, and increased sensitivity to stress. Our data suggest that N-methylation of Rpt1 and/or its PK sequence might be important in cell growth or stress tolerance in yeast.
26S 蛋白酶体是一种多催化蛋白酶复合物，可降解真核细胞中泛素化的蛋白质。它由一个蛋白水解核心（20S 蛋白酶体）以及调节颗粒组成，后者包含六个 ATP 酶（Rpt）亚基，参与底物的展开和向 20S 蛋白酶体的催化腔的易位。在这项研究中，我们使用 MS 分析了酵母 Rpt1 亚基的 N 末端修饰，该亚基包含 N-甲基转移酶的 N 末端识别序列。我们的结果表明，在去除酵母 Rpt1 的起始 Met 残基后，N 末端 Pro 残基要么未修饰，要么单甲基化或二甲基化，并且这种甲基化在进化过程中没有保守。为了更好地了解酵母 Rpt1 第 3 和第 4 位的 Pro-Lys（PK）序列的可能功能，我们生成了表达缺失该序列的 Rpt1 等位基因的突变株。PK 序列的缺失会导致 N-甲基化缺失、细胞生长减少和对压力的敏感性增加。我们的数据表明，Rpt1 的 N-甲基化和/或其 PK 序列可能在酵母的细胞生长或应激耐受中很重要。
