NIH RESOURCE PROTEIN ANALYSIS

University Of Washington, Seattle WA

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Abstract

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 There is unpublished biochemical evidence that both YDR069c and YDL020c associate, but it is not clear what the stoichiometry of association is relative to other subunits.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.