Doa1 is a Cdc48 adapter that possesses a novel ubiquitin binding domain.
Cdc48 (p97/VCP) is an AAA-ATPase molecular chaperone whose cellular functions are facilitated by its interaction with ubiquitin binding cofactors (e.g., Npl4-Ufd1 and Shp1). Several studies have shown that Saccharomyces cerevisiae Doa1 (Ufd3/Zzz4) and its mammalian homologue, PLAA, interact with Cdc48. However, the function of this interaction has not been determined, nor has a physiological link between these proteins been demonstrated. Herein, we demonstrate that Cdc48 interacts directly with the C-terminal PUL domain of Doa1. We find that Doa1 possesses a novel ubiquitin binding domain (we propose the name PFU domain, for PLAA family ubiquitin binding domain), which appears to be necessary for Doa1 function. Our data suggest that the PUL and PFU domains of Doa1 promote the formation of a Doa1-Cdc48-ubiquitin ternary complex, potentially allowing for the recruitment of ubiquitinated proteins to Cdc48. DOA1 and CDC48 mutations are epistatic, suggesting that their interaction is physiologically relevant. Lastly, we provide evidence of functional conservation within the PLAA family by showing that a human-yeast chimera binds to ubiquitin and complements doa1Delta phenotypes in yeast. Combined, our data suggest that Doa1 plays a physiological role as a ubiquitin binding cofactor of Cdc48 and that human PLAA may play an analogous role via its interaction with p97/VCP.
Mullally
James E
JE
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
Chernova
Tatiana
T
Wilkinson
Keith D
KD
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D000074405:Valosin Containing Protein
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C099944:DOA1 protein, S cerevisiae; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C055324:phospholipase A2-activating protein; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; C000614178:VCP protein, human; D000074405:Valosin Containing Protein