Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase.
To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1. That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.
Fransen
M
M
Department of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA.
Van Veldhoven
P P
PP
Subramani
S
S
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D000074409:Carbonyl Reductase (NADPH); D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008830:Microbodies / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D000074409:Carbonyl Reductase (NADPH); C095961:DHRS2 protein, human; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase