Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily.
A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd approximately 0.37 microM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.
Weber
F E
FE
Klinik für Gesichts-und Kieferchirurgie, Universitätsspital Zürich, Frauenklinikstrasse 10, 8091 Zurich, Switzerland. weber@zzmk.unizh.ch
Minestrini
G
G
Dyer
J H
JH
Werder
M
M
Boffelli
D
D
Compassi
S
S
Wehrli
E
E
Thomas
R M
RM
Schulthess
G
G
Hauser
H
H
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D006801:Humans; D007150:Immunohistochemistry; D007421:Intestine, Small / Q000378:metabolism / Q000648:ultrastructure; D008830:Microbodies / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D011817:Rabbits
D002352:Carrier Proteins; D002118:Calcium