RGD Reference Report - Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase.

General

Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase.
Authors:	Fransen, M Van Veldhoven, PP Subramani, S
Citation:	Fransen M, etal., Biochem J 1999 Jun 1;340 ( Pt 2):561-8.
RGD ID:	70713
Pubmed:	PMID:10333503 (View Abstract at PubMed)
PMCID:	PMC1220285 (View Article at PubMed Central)
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.

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Cellular Component

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Term	Qualifier	Evidence	With	Reference	Notes	Source	Original Reference(s)
peroxisomal membrane	located_in	IDA		70713	PMID:10333503	UniProt
peroxisome		IDA		70713		RGD

Molecular Function

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Term	Qualifier	Evidence	With	Reference	Notes	Source	Original Reference(s)
2,4-dienoyl-CoA reductase (NADPH) activity		IDA		70713		RGD

Objects Annotated

Genes (Rattus norvegicus)

Decr2 (2,4-dienoyl-CoA reductase 2)

Dhrs4 (dehydrogenase/reductase 4)

Objects referenced in this article

Gene	DHRS4	dehydrogenase/reductase 4	Homo sapiens
Gene	DHRS4L1	dehydrogenase/reductase 4 like 1 (pseudogene)	Homo sapiens

Additional Information

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Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase.