Abstract
2-Methylacyl-CoA racemase is an auxiliary enzyme required for the peroxisomal β-oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer of C27 bile acid intermediates. The enzyme activity is found not only in peroxisomes but also is present in mitochondria of human liver and fibroblasts. The C terminus of the human racemase, a protein of 382 amino acids with a molecular mass of 43,304 daltons as deduced from its cloned cDNA, consists of KASL. Hitherto this sequence has not been recognized as a peroxisomal targeting signal (PTS1). From the in vitro interaction between recombinant racemase and recombinant human PTS1 receptor (Pex5p), and the peroxisomal localization of green fluorescent protein (GFP) fused to the N terminus of full-length racemase or its last six amino acids in tranfected Chinese hamster ovary (CHO) cells, we concluded that ASL is a new PTS1 variant. To be recognized by Pex5p, however, the preceding lysine residue is critical. As shown in another series of transfection experiments with GFP fused to the C terminus of the full-length racemase or racemase with deletions of the N terminus, mitochondrial targeting information is localized between amino acids 22 and 85. Hence, our data show that a single transcript gives rise to a racemase protein containing two topogenic signals, explaining the dual cellular localization of the activity.—Amery, L., M. Fransen, K. De Nys, G. P. Mannaerts, and P. P. Van Veldhoven. Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans. J. Lipid Res. 2000. 41: 1752–1759.
Highlights
2-Methylacyl-CoA racemase is an auxiliary enzyme required for the peroxisomal -oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer of C27 bile acid intermediates
On the basis of the rat racemase amino acid sequences [13, 14], expressed sequence tag (EST) of the corresponding human protein were retrieved from the databases
The cDNA of the human 2methylacyl-CoA racemase,3 composed from overlapping sequences present in the IMAGE [18] EST clone 788810 and 5Ј-rapid amplification of cDNA ends (RACE) products, obtained from a gt11 library, contained an open reading frame of 1,149 bases encoding a protein of 42,304 Da (Fig. 1)
Summary
To obtain the human racemase cDNA sequence, we used the rat racemase/epimerase amino acid sequence [13, 14] as a query to search [17] the expressed sequence tag (EST) database for corresponding human ESTs. To perform interaction studies with the PTS1 receptor Pex5p, a 600-bp EcoRI fragment of clone 788180 was inserted into EcoRItreated pGEX-4T-1 (Amersham Pharmacia, Piscataway, NJ), creating a vector encoding glutathione S-transferase (GST) fused to the last 157 amino acids of human racemase. This ligation mixture was used to transform Escherichia coli DH5␣ cells and clones containing the insert in the correct orientation were selected. The 1.16-kb specific PCR product was gel purified, digested with BglII and SalI, and subcloned into the BamHI/SalI-digested two-hybrid system bait vector pGBT9 (Clontech) and the BamHI/SalI-digested pQE32 expression vector (Qiagen, Valencia, CA) These constructs, pMF282 and pMF323, encode Gal4bd-HsRacemase and hexahistidine (His)6HsRacemase, respectively. Racemase activities were measured in lysates of E. coli Top10FЈ expressing the (His)6-HsRacemase or GST-HsRacemase as described previously [9]
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