Nucleolar localization of cirhin, the protein mutated in North American Indian childhood cirrhosis

Cirhin (NP_116219), the product of the CIRH1A gene is mutated in North American Indian childhood cirrhosis (NAIC/CIRH1A, OMIM 604901), a severe autosomal recessive intrahepatic cholestasis. It is a 686-amino-acid WD40-repeat containing protein of unknown function that is predicted to contain multiple targeting signals, including an N-terminal mitochondrial targeting signal, a C-terminal monopartite nuclear localization signal (NLS) and a bipartite nuclear localization signal (BNLS). We performed the direct determination of subcellular localization of cirhin as a crucial first step in unraveling its biological function. Using EGFP and His-tagged cirhin fusion proteins expressed in HeLa and HepG2, cells we show that cirhin is a nucleolar protein and that the R565W mutation, for which all NAIC patients are homozygous, has no effect on subcellular localization. Cirhin has an active C-terminal monopartite nuclear localization signal (NLS) and a unique nucleolar localization signal (NrLS) between residues 315 and 432. The nucleolus is not known to be important specifically for intrahepatic cholestasis. These observations provide a new dimension in the study of hereditary cholestasis.