Proteomic Analysis of Dhh1 Complexes Reveals a Role for Hsp40 Chaperone Ydj1 in Yeast P-Body Assembly

Abstract

Abstract P-bodies (PB) are ribonucleoprotein (RNP) complexes that aggregate into cytoplasmic foci when cells are exposed to stress. Although the conserved mRNA decay and translational repression machineries are known components of PB, how and why cells assemble RNP complexes into large foci remain unclear. Using mass spectrometry to analyze proteins immunoisolated with the core PB protein Dhh1, we show that a considerable number of proteins contain low-complexity sequences, similar to proteins highly represented in mammalian RNP granules. We also show that the Hsp40 chaperone Ydj1, which contains an low-complexity domain and controls prion protein aggregation, is required for the formation of Dhh1−GFP foci on glucose depletion. New classes of proteins that reproducibly coenrich with Dhh1−GFP during PB induction include proteins involved in nucleotide or amino acid metabolism, glycolysis, transfer RNA aminoacylation, and protein folding. Many of these proteins have been shown to form foci in response to other stresses. Finally, analysis of RNA associated with Dhh1−GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins. Thus, global characterization of PB composition has uncovered proteins important for PB assembly and evidence suggesting an active role for RNA in PB function.