Molecular chaperone GRP94 binds to the Fanconi anemia group C protein and regulates its intracellular expression.
The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence of FAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.
Hoshino
T
T
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Wang
J
J
Devetten
M P
MP
Iwata
N
N
Kajigaya
S
S
Wise
R J
RJ
Liu
J M
JM
Youssoufian
H
H
D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D005199:Fanconi Anemia / Q000378:metabolism*; D052218:Fanconi Anemia Complementation Group C Protein; D051856:Fanconi Anemia Complementation Group Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014162:Transfection
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C497652:Fancc protein, mouse; C497653:Fancc protein, rat; D052218:Fanconi Anemia Complementation Group C Protein; D051856:Fanconi Anemia Complementation Group Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011506:Proteins; C018551:glucose-regulated proteins; D016685:Mitomycin