Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23.
Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates. The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation. The major function of Ub is to regulate the in vivo half-life of its substrates. Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it. Molecular mechanisms of the postubiquitylation processes are poorly understood. Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed the ubiquitin-associated domain (UBA). Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation. We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis. Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome. Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events.
Rao
Hai
H
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. raoh@cco.caltech.edu
Sastry
Ashwani
A
D020816:Amino Acid Motifs; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C100415:DSK2 protein, S cerevisiae; D005656:Fungal Proteins; C490846:RAD23 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D014452:Ubiquitins; D005982:Glutathione Transferase; D008239:Lysine