Mechanistic insight into site-restricted monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI.
A key step in the Fanconi anemia (FA) tumor suppressor pathway is the site-specific monoubiquitination of the FANCD2 protein. Genetic studies indicate that this crucial modification requires eight known FA gene products and the E2-conjugating enzyme Ube2t. Here, we minimally reconstitute this monoubiquitination reaction with Ube2t and the FANCL protein, revealing that monoubiquitination is stimulated by a conserved RWD-like domain in FANCL. Furthermore, addition of the FANCI protein enhances monoubiquitination and also restricts it to the in vivo substrate lysine residue on FANCD2. This work therefore establishes a system that provides mechanistic insight into the functions of FANCL and FANCI in the catalysis of FANCD2 monoubiquitination.
Alpi
Arno F
AF
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Pace
Paul E
PE
Babu
M Madan
MM
Patel
Ketan J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D017124:Conserved Sequence; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D052240:Fanconi Anemia Complementation Group L Protein / Q000737:chemistry / Q000378:metabolism*; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D054875:Ubiquitination
C519921:FANCI protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins; C513404:UBE2T protein, human; C510626:UBE2W protein, human; D044763:Ubiquitin-Conjugating Enzymes; D052240:Fanconi Anemia Complementation Group L Protein