Identification of cytosolic proteins that bind to the Fanconi anemia complementation group C polypeptide in vitro. Evidence for a multimeric complex.
The oligomeric structure of Fanconi anemia complementation group C (FACC) was investigated in mammalian cell lysates. Using an affinity-purified polyclonal antibody, FACC was immunoprecipitated from radiolabeled cell lysates and shown to form monomers of 63 kDa. Association of FACC with heterologous proteins was investigated by co-precipitation of radiolabeled proteins with a recombinant chimeric FACC molecule fused to the constant portion of the human IgG1 heavy chain (FACC gamma 1). Expression of FACC gamma 1 in FACC-deficient Fanconi anemia (FA) lymphoblasts corrected the hypersensitivity of these cells to mitomycin C. Binding of FACC gamma 1 to protein A-agarose and incubation with radiolabeled cell lysates identified three polypeptides with molecular masses of 65, 50, and 35 kDa that were also detected on immunoblots probed with the purified FACC gamma 1 polypeptide. FACC, as well as the three FACC-binding polypeptides, co-fractionated with cytosolic and membrane extracts. Binding was specific for the FACC moiety of FACC gamma 1 and was detected in cytosolic extracts of a number of FA and non-FA mammalian cells. These results demonstrate that FACC binds directly to a family of ubiquitous cytosolic proteins and is conserved in a wide range of mammalian cells.
Youssoufian
H
H
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Auerbach
A D
AD
Verlander
P C
PC
Steimle
V
V
Mach
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005199:Fanconi Anemia / Q000378:metabolism*; D052218:Fanconi Anemia Complementation Group C Protein; D051856:Fanconi Anemia Complementation Group Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010455:Peptides / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C497651:FANCC protein, human; D052218:Fanconi Anemia Complementation Group C Protein; D051856:Fanconi Anemia Complementation Group Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins