An Armadillo motif in Ufd3 interacts with Cdc48 and is involved in ubiquitin homeostasis and protein degradation.
The yeast AAA-ATPase Cdc48 and the ubiquitin fusion degradation (UFD) proteins play important, evolutionarily conserved roles in ubiquitin dependent protein degradation. The N-terminal domain of Cdc48 interacts with substrate-recruiting cofactors, whereas the C terminus of Cdc48 binds to proteins such as Ufd3 that process substrates. Ufd3 is essential for efficient protein degradation and for maintaining cellular ubiquitin levels. This protein contains an N-terminal WD40 domain, a central ubiquitin-binding domain, and a C-terminal Cdc48-binding PUL domain. The crystal structure of the PUL domain reveals an Armadillo repeat with high structural similarity to importin-alpha, and the Cdc48-binding site could be mapped to the concave surface of the PUL domain by biochemical studies. Alterations of the Cdc48 binding site of Ufd3 by site-directed mutagenesis resulted in a depletion of cellular ubiquitin pools and reduced activity of the ubiquitin fusion degradation pathway. Therefore, our data provide direct evidence that the functions of Ufd3 in ubiquitin homeostasis and protein degradation depend on its interaction with the C terminus of Cdc48.
Zhao
Gang
G
Center for Structural Biology and Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA.
Li
Guangtao
G
Schindelin
Hermann
H
Lennarz
William J
WJ
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D051186:Armadillo Domain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005470:Fluorometry; D006706:Homeostasis; D006868:Hydrolysis; D015151:Immunoblotting; D047468:Immunoprecipitation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000378:metabolism*; D000074405:Valosin Containing Protein
D048868:Adaptor Proteins, Signal Transducing; D051186:Armadillo Domain Proteins; D018797:Cell Cycle Proteins; C099944:DOA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein