To date, isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase; EC 5.3.3.2) is presumed to have a cytosolic localization. However, we have recently shown that in permeabilized cells lacking cytosolic components, mevalonate can be converted to cholesterol, implying that all of the enzymes required for the conversion of mevalonate to farnesyl diphosphate are found in the peroxisome. To provide unequivocal evidence for the subcellular localization of IPP isomerase, in this study, we have cloned the rat and hamster homologues of IPP isomerase and identified the signal that targets this enzyme to peroxisomes. In addition, we also demonstrate that IPP isomerase is regulated at the mRNA level.The isoprenoid biosynthetic pathway is ubiquitous to all living organisms. A few of the important end products of this complex pathway include: dolichols; vitamins A, D, E, and K; steroid hormones; carotenoids; bile acids; and cholesterol (1). The enzyme isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase 1 ; EC 5.3.3.2) plays a crucial role in this pathway by catalyzing the interconversion of isopentenyl diphosphate (IPP) to its highly electrophilic isomer, dimethylallyl diphosphate (2). These two isomers are the building blocks for the successive head-to-tail condensation reactions that result in the synthesis of farnesyl diphosphate (FPP), and ultimately, cholesterol (3).Recently, it has been shown by our group and others that peroxisomes contain a number of enzymes involved in cholesterol biosynthesis that were previously thought to be cytosolic. Specifically, peroxisomes have been shown to contain acetoacetyl-CoA thiolase (4, 5), 3-hydroxy-3-methylglutaryl coenzyme A synthase (6), 3-hydroxy-3-methylglutaryl coenzyme A reductase (7-9), mevalonate kinase (10, 11), phosphomevalonate kinase (12), mevalonate diphosphate decarboxylase (12), and FPP synthase (13). Both mevalonate kinase and FPP synthase seem to be localized predominantly, if not exclusively, to peroxisomes (11, 13). To date, IPP isomerase is presumed to have a cytosolic localization (1); however, the following three observations have led us to believe that the enzyme is localized to peroxisomes: (i) in permeabilized cells lacking cytosolic components, mevalonate can be converted to cholesterol in equal amounts to that observed in nonpermeabilized cells, therefore suggesting that the cytosol does not contain enzymes necessary for the conversion of mevalonate to FPP (12); (ii) IPP isomerase activity in tissues from patients with peroxisome-deficient diseases (Zellweger and neonatal adrenoleukodystrophy) is 50% of that found in tissues from control patients (13); and (iii) the deduced amino acid sequence from the human isomerase cDNA, which has been recently cloned (14) and characterized (15), contains two putative peroxisomal targeting sequences.At the C-terminal end of human isomerase is a putative peroxisomal targeting sequence 1 (PTS1) consisting of YRM (single-letter amino acid notation), and at the N-terminal end is a p...

In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study.In mammalian cells, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 1 reductase is the rate-limiting enzyme for the synthesis of mevalonic acid, the precursor of cholesterol and other non-sterol isoprenoids. We and others (1-4) have demonstrated that HMG-CoA reductase is localized in two distinct intracellular compartments, endoplasmic reticulum (ER) and peroxisomes. ER HMG-CoA reductase is a 97-kDa transmembrane glycoprotein. A short non-conserved sequence links the multiple transmembrane domain to the highly conserved catalytic domain, which extends out into the cytosol. Because of its role in cholesterol biosynthesis, the regulation of HMG-CoA reductase has been intensely studied. The levels of the ER enzyme are regulated by transcription (5-7), translation (8, 9), and enzyme degradation (10, 11). Another critical role for this enzyme has emerged in recent years, due to the requirement of farnesyl diphosphate and geranyl-geranyl diphosphate in isoprenylation of proteins (12). Keller et al. (1) were the first to demonstrate that in the liver HMG-CoA reductase is present not only in the ER but also within the peroxisomes. The function of the peroxisomal reductase in cholesterol/isoprenoid metabolism has yet to be defined. However, it is clear that the ER and peroxisomal HMG-CoA reductases can be regulated differently and, therefore, may play different functional roles (2, 13). The ER reductase has a diurnal cycle distinct from that of the peroxisomal reductase (13). However, the two reductases can also be regulated coordinately. Both reductase activities are induced by cholestyramine (a bile acid resin) (2). No information is available regarding the function of the peroxisomal reductase in cholesterol/ isoprenoid metabolism, nor has the structure of the peroxisomal HMG-CoA reductase been determined. Accordingly, to facilitate our studies of the f...

Our group and others have recently demonstrated that peroxisomes contain a number of enzymes involved in cholesterol biosynthesis that previously were considered to be cytosolic or located in the endoplasmic reticulum (ER). Peroxisomes have been shown to contain HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl diphosphate isomerase, and FPP synthase. Four of the five enzymes required for the conversion of mevalonate to FPP contain a conserved putative PTS1 or PTS2, supporting the concept of targeted transport into peroxisomes. To date, no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein, and which is localized exclusively to peroxisomes, to facilitate our studies on the function, regulation, and structure of the peroxisomal HMG-CoA reductase. This cell line was obtained by growing UT2 cells (which lack the ER HMG-CoA reductase) in the absence of mevalonate. The surviving cells exhibited a marked increase in a 90-kD HMG-CoA reductase that was localized exclusively to peroxisomes. The wild-type CHO cells contain two HMG-CoA reductase proteins, the well-characterized 97-kD protein localized in the ER, and a 90-kD protein localized in peroxisomes. We have also identified the mutations in the UT2 cells responsible for the lack of the 97-kD protein. In addition, peroxisomal-deficient Pex2 CHO cell mutants display reduced HMG-CoA reductase levels and have reduced rates of sterol and nonsterol biosynthesis. These data further support the proposal that peroxisomes play an essential role in isoprenoid biosynthesis.

UT2 cells are a mutant clone of Chinese hamster ovary (CHO) cells that are deficient in the 97 kDa endoplasmic reticulum (ER) 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein. The analysis of UT2 cell cDNA and genomic DNA has led to the identification of two novel point mutations in intronic sequences of the ER HMG-CoA reductase gene. One mutation identified at the ؉ 1 position (G → A) of the 5 splice site of exon 11-12 junction was shown to cause exon 11 skipping which resulted in the insertion of premature stop codons. We also identified a second mutation at the ؉ 5 position (G → A) of the 5 splice site in the intron spanning exons 13 and 14. Furthermore, the data indicate that the two mutations in the reductase gene are present on the same allele. As demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) of UT2 cell mRNA, the mutations produce aberrant spliced messages. If the aberrant messages were translated, truncated proteins of 44 kDa or 66 kDa would be predicted. More importantly, these truncated proteins would be expected not to have catalytic activity. In addition, we have also recently demonstrated that the UT2 cells express a 90 kDa HMG-CoA reductase protein that is localized exclusively in peroxisomes, and is up-regulated when the cells are grown in the absence of added mevalonate. Thus, the mutations identified in the ER reductase gene in UT2 cells indicate that neither a 97 kDa nor a 90 kDa reductase protein can be produced from this gene.-Engfelt, W. H., K. R. Masuda, V. G. Paton, and S. K. Krisans. Splice donor site mutations in the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene cause a deficiency of the endoplasmic reticulum 3-hydroxy-3-methylglutaryl coenzyme A reductase protein in UT2 cells.

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