Role of peroxisomes in isoprenoid biosynthesis.

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.

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.

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.

Characterization of UT2 cells. The induction of peroxisomal 3-hydroxy-3-methylglutaryl-coenzyme a reductase.

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.