In mouse alpha -methylacyl-CoA racemase, the same gene product is simultaneously located in mitochondria and peroxisomes.
alpha-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid degradation pathway, was studied at the protein, cDNA, and genomic levels in mouse liver. Immunoelectron microscopy and subcellular fractionation located racemase to mitochondria and peroxisomes. The enzymes were purified from both organelles with immunoaffinity chromatography. The isolated proteins were of the same size, with identical N-terminal amino acid sequences, and the existence of additional proteins with alpha-methylacyl-CoA racemase activity was excluded. A racemase gene of about 15 kilobases was isolated. Southern blot analysis and chromosomal localization showed that only one racemase gene is present, on chromosome 15, region 15B1. The putative initial ATG in the racemase gene was preceded by a functional promotor as shown with the luciferase reporter gene assay. The corresponding cDNAs were isolated from rat and mouse liver. The recombinant rat protein was overexpressed in active form in Pichia pastoris. The presented data suggest that the polypeptide encoded by the racemase gene can alternatively be targeted to peroxisomes or mitochondria without modifications. It is concluded that the noncleavable N-terminal sequence of the polypeptide acts as a weak mitochondrial and that the C-terminal sequence acts as a peroxisomal targeting signal.
Kotti
T J
TJ
Biocenter Oulu and Department of Biochemistry, University of Oulu, Linnanmaa, Oulu FIN-90014, Finland.
Savolainen
K
K
Helander
H M
HM
Yagi
A
A
Novikov
D K
DK
Kalkkinen
N
N
Conzelmann
E
E
Hiltunen
J K
JK
Schmitz
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005091:Exons; D017930:Genes, Reporter; D007438:Introns; D051379:Mice; D016253:Microscopy, Immunoelectron; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D020675:Peroxisomes / Q000201:enzymology* / Q000648:ultrastructure; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000378:metabolism; D019998:Racemases and Epimerases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment
D021382:Protein Sorting Signals; D012333:RNA, Messenger; D019998:Racemases and Epimerases; C108352:alpha-methylacyl-CoA racemase