NIH RESOURCE PROTEIN ANALYSIS

酿酒酵母蛋白酶体的分离和鉴定 使用LC/MS/MS鉴定成分。 蛋白酶体已分离到19S和26S复合体。在26S中 亚基，共鉴定出29种蛋白质，其中27种 可以被指定为蛋白酶体亚基。已知的14个20岁中的12个 确定了亚基，17个预测的帽亚单位中有15个亚基 都是在19S年代的分析中确定的。两种蛋白质中不能 被分配，1具有未知的功能，并在19世纪90年代被发现 也做好准备。另一种蛋白质是Med8，已被鉴定为 多亚基RNA聚合酶II介体的一个亚基。 来自19s复合体的数据包含17个已确认的序列 和推定的19S亚基。在MIPS蛋白质复合体数据库中， 他们列出了18个“预测”的19亚基。我们已经确认了其中15个 这些，外加两个不在MIPS列表中(但这两个是 YPD基于同源性预测的蛋白酶体亚基 哺乳动物蛋白质)。我们没有找到的3个是YGR270W、YDR069c和 YDL020c.其中，几乎没有理由相信YGR270W是 一个真正的亚单位。有未发表的生化证据表明 YDR069c和YDL020c关联，但尚不清楚 缔合的化学计量学是相对于其他亚基的。它是 值得注意的是，对于质谱仪鉴定的6个亚基，有 没有先前的生化或遗传数据来支持他们被指定为 蛋白酶体亚基。数据库分配完全基于 关于克隆的哺乳动物亚基的同源性。这些基因中没有一个是 至少根据YPD的说法，它还没有被淘汰。下一堂课 鉴定的蛋白质为热休克蛋白。有证据表明， 文献表明HSP70家族成员以某种方式参与蛋白质 营业额。此外，由于热休克蛋白与变性蛋白结合，而 蛋白酶体降解变性的蛋白质，这是有意义的，两者 可能会相互作用(尽管可能是间接的)。最后一节课 蛋白质是未知的/特征不佳的/杂的。其中一些人 耐人寻味的是：Mpt4抑制了Hect结构域蛋白中的突变 (Hect结构域=Ub连接酶)；Trx2与Sec18在功能上相互作用， 它是与蛋白酶体ATPase相关的AAA ATPase；Bmh1，2是 信号转导调节因子Dbp9和Sub2是可能的解旋酶 (19S帽的一个真正的ATPase亚基Sug1/Cim3具有 被证明具有解旋酶活性)；CDC48是必需的AAA ATPase 泛素融合蛋白的周转。

The proteasome of S. cerevisiae has been isolated and the components are being identified using LC/MS/MS. Two forms of the proteasome have been isolated the 19S and 26S complexes. In the 26S subunit, 29 proteins have been identified by mass spec, 27 of which can be assigned as proteasome subunits. 12 of the 14 known 20S subunits were identified, and 15 of the 17 predicted cap subunits that were identified in the 19S analysis. Of the two proteins that can't be assigned, 1 is of unknown function, and was identified in the 19S prep as well. The other protein is Med8, which has been identified as a subunit of the multisubunit RNA polymerase II mediator. The data from the 19S complex contain sequences from 17 confirmed and putative 19S subunits. In the MIPS database on protein complexes, they list 18 "predicted" 19S subunits. We have identified 15 of these, plus two that are not in the MIPS list (but these 2 are predicted by YPD to be proteasome subunits based on homology to mammalian proteins). The 3 we didn't find are YGR270W, YDR069c, and YDL020c. Of these, there is little reason to believe that YGR270W is a true subunit. There is unpublished biochemical evidence that both YDR069c and YDL020c associate, but it is not clear what the stoichiometry of association is relative to other subunits. It is worth noting that for 6 of the subunits identified by mass spec, there is no prior biochemical or genetic data to support their assignment as proteasomal subunits. The database assignments were made based solely on homology to cloned mammalian subunits. None of these genes has been knocked out yet, at least according to YPD. The next class of proteins identified is heat shock protein. There is evidence in the literature that HSP70 family members are somehow involved in protein turnover. Moreover, since HSPs bind denatured proteins, and the proteasome degrades denatured proteins, it make sense that the two might interact (albeit, possibly indirectly). The last class of proteins is unknown/poorly characterized/miscellaneous. Some of them are intriguing: Mpt4 suppresses a mutant in a HECT domain protein (HECT domain = Ub ligase); Trx2 interacts functionally with Sec18, which is an AAA ATPase related to the proteasomal ATPases; Bmh1,2 are signal transduction regulators, Dbp9 and Sub2 are putative helicases (one of the bona fide ATPase subunits of the 19S cap, Sug1/Cim3, has been shown to have helicase activity); Cdc48 is an AAA ATPase required for the turnover of ubiquitin fusion proteins.