Evidence for a Menkes-like protein with a nuclear targeting sequence.
Extracts from three human cell lines were found to contain abridged Menkes disease gene transcripts with novel insertion sequences. The transcript variant that is the focus of the present study codes for a 103-residue protein containing the first heavy-metal-binding domain (Hmb1) of ATP7A, the Cu-ATPase associated with Menkes disease. This transcript variant has a 45-bp nucleotide insert interposed between exons 1 and 2 of ATP7A that starts with a 5' ATG that is in-frame with the downstream ATG translation start site of ATP7A. We report here that the 66-bp nucleotides positioned between the upstream and downstream ATG sites encode 22 amino acid residues whose primary structure in part meets the criteria for a nuclear-localization sequence (NLS). We have referred to the transcript as nuclear Menkes-like (NML) 45. A green fluorescent protein (GFP) construct with NML45 when transfected in Chinese hamster ovary cells localized to the cell nucleus. A similar construct without the 66-bp segment exhibited a random dispersed fluorescent pattern in the cytosol. GFP constructs encoding ATP7A exons likewise failed to direct GFP into the cell nucleus, suggesting the nuclear determinant is not in an internal domain of the protein. The data suggest that the 22-residue segment contains an NLS for an 11.2-kDa protein with one Cu-binding site that may function as a chaperone to transport Cu into the nucleus of mammalian cells.
Reddy
M C
MC
Department of Biochemistry and Biophysics and the Faculty of Nutrition, Texas A&M University, 2128 TAMUS, College Station, TX 77843-2128, USA.
Majumdar
S
S
Harris
E D
ED
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D000073840:Copper-Transporting ATPases; D006224:Cricetinae; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D018076:DNA, Complementary; C418185:NML45 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases