Symposium Papers |
Correspondence to: Skaidrite K. Krisans, Dept. of Biology, San Diego State Univ., San Diego, CA 92182.
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Summary |
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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. (J Histochem Cytochem 47:11271132, 1999)
Key Words: peroxisomes, isoprenoids, cholesterol, HMG-CoA reductase
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Introduction |
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The isoprenoid biosynthetic pathway is ubiquitous to all living organisms. A few of the important endproducts of this complex pathway include dolichols, vitamins A, D, E, and K, steroid hormones, carotenoids, bile acids, and cholesterol.
Recent studies by our group and others have demonstrated that a number of the enzymes of the isoprenoid biosynthetic pathway are localized to peroxisomes (
The peroxisomal enzymes required for conversion of mevalonate to FPP (i.e., mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isopentenyl diphosphate isomerase, and FPP synthase) have now been cloned and sequenced. Four of the five enzymes, mevalonate kinase (
Until recently, IPP isomerase was presumed to have a cytosolic localization. However, the following three observations led us to believe that the enzyme may be localized to peroxisomes. (a) In permeabilized cells lacking cytosolic components, mevalonate can be converted to cholesterol in equal amounts to those observed in nonpermeabilized cells, this suggesting that the cytosol does not contain enzymes necessary for the conversion of mevalonate to FPP (
At the C-terminal end of human isomerase is a putative PTS1 consisting of YRM (single-letter amino acid notation) and at the N-terminal end is a putative PTS2 sequence consisting of HLX5QL (where X designates any amino acid). The consensus sequence for the PTS1 motif is (S/A/C)(K/H/R)(L/M) (
We have cloned the rat and hamster homologues of IPP isomerase and have demonstrated that the protein is targeted to peroxisomes by use of the PTS1 motif (
To determine the subcellular compartment in which the IPP isomerase is localized, we transfected the full-length construct into CHO cells. The cells were then simultaneously immunolabeled with anti-catalase antibody (Figure 1A) and anti-HA antibody (Figure 1B). The immunofluorescence pattern for catalase was superimposable over the pattern obtained with the HA antibody. Similar results were obtained in human fibroblast cells labeled with anti-catalase antibody and anti-HA antibody (
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To determine which of the PTS targeting signals are utilized by the hamster isomerase, we utilized two distinct cell lines derived from patients with peroxisomal disorders. One cell line was shown to be deficient in the peroxisomal import of only PTS2 proteins; the other cell line was shown to be deficient in the peroxisomal import of both PTS1 and PTS2 proteins. To first determine if the putative PTS2 is responsible for peroxisomal import, the full-length construct was transfected into the PTS2-deficient cell line. The cells were again double-labeled with anti-catalase antibody and anti-HA antibody. The data again demonstrated a superimposable punctate pattern when labeling with anti-catalase and anti-HA antibodies was performed. These results suggest that IPP isomerase does not use the putative PTS2 for peroxisomal targeting.
To provide direct evidence that the putative PTS1 of isomerase is necessary for peroxisomal targeting, a second expression vector was constructed in which the HRM tripeptide was deleted. This deletion construct was transfected into the PTS2-deficient cell line. The immunofluorescence pattern was consistent with peroxisomal labeling when anti-catalase antibody was used (Figure 1C), whereas a cytosolic labeling pattern was obtained when the anti-HA antibody was used (Figure 1D). These data therefore show that the HRM tripeptide is necessary for the targeting of IPP isomerase to peroxisomes.
The HRM tripeptide is present at the C-terminus of both the rat and the hamster homologue of IPP isomerase. However, the human homologue has a YRM tripeptide at its C-terminus (
The PTS1 consensus sequence of (S/A/C)(K/H/R) (L/M) was derived from extensive mutational analysis where peroxisomal proteins from other organisms or nonperoxisomal proteins were used as reporters. Since the formulation of this consensus sequence, more peroxisomal proteins have been identified whose PTS1-like tripeptide does not fit this exact sequence. However, it has been recently demonstrated that many amino acid substitutions can be made at the first position of a homologous protein without compromising the PTS1 function (
In addition to the above-mentioned enzymes, four of the enzyme activities (dihydrolanosterol oxidase, steroid-14-reductase, steroid-3-ketoreductase, and steroid-8-isomerase) involved in the conversion of lanosterol to cholesterol have also been reported to be present in peroxisomes (
Figure 2 illustrates our current concept of the compartmentalization of cholesterol biosynthetic enzymes (
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The indispensable role of the peroxisomal enzymes in isoprenoid biosynthesis is also evident in humans afflicted by the recessive inherited peroxisomal disorders (PDs), e.g., Zellweger syndrome (ZS), in which normal peroxisomes are absent from the cells (
The localization of this protein to peroxisomes was demonstrated by four different methods: (a) analytical subcellular fractionation and measurement of enzyme activities; (b) immunoblotting for HMG-CoA reductase in the isolated fractions with a monospecific antibody; (c) immunofluoresence microscopy; and (d) immunoelectron microscopy. All four methods produced consistent results. The conclusion that the 90-kD protein localized in peroxisomes is HMG-CoA reductase is based on the following findings (a) A number of different monospecific HMG-CoA reductase antibodies crossreact with this protein. (b) The proteins were specifically precipitated as they were competed by an excess of the corresponding free peptides. (c) The HMG-CoA reductase antibody specifically immunoprecipitated the HMG-CoA reductase activity. (d) The protein and HMG-CoA reductase activity levels are regulated coordinately. Finally, (e) the HMG-CoA reductase activity is completely abolished in vitro by addition of lovastatin.
We have recently determined that the deficiency of the 97-kD ER HMG-CoA reductase protein in UT2 cells is caused by two mutations within the gene (
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From these data, it is clear that the UT2 cells produce aberrantly spliced HMG-CoA reductase transcripts unable to code for a 97- or 90-kD reductase protein. In addition, we have never obtained alternatively spliced messages capable of coding for a 90-kD protein by use of RT-PCR analysis of CHO or UT2/UT2* cell ER reductase mRNA or by screening of UT2* cell cDNA libraries with the full-length reductase probe. Therefore, these data suggest that the 90-kD HMG-CoA reductase found in CHO and UT2/UT2* cells may be a product of a novel gene.
Our hypothesis is that all wild-type cells contain two forms of HMG-CoA reductase. The UT2 cells lack the ER HMG-CoA reductase as a result of chemical mutagenesis (
In mammals, only one gene has been found to encode HMG-CoA reductase. However yeast, fungi, and plants all contain more than one HMG-CoA reductase gene. Yeast, fungi and Arabidopsis thaliana all contain two genes (
We have also performed a detailed analysis of the isoprenoid biosynthetic pathway in the peroxisome-deficient CHO cell lines ZR-78 and ZR-82 (
The results showed that total HMG-CoA reductase activity was significantly reduced in the peroxisome-deficient cells compared to the wild-type cells. Analysis of the two reductase proteins in permeabilized cells indicated that, in the ZR-78 and ZR-82 cells, the 90-kD peroxisomal reductase protein was mainly localized to the cytosol. In addition, the rates of both sterol (cholesterol) and nonsterol (dolichols) biosynthesis were significantly lower in the peroxisome-deficient cells when either acetate or mevalonate was used as substrate. In contrast, the rate of dolichol biosynthesis in the peroxisome-deficient cells was similar to that of the wild-type cells when incubated with farnesol. Furthermore, the data also indicated that the peroxisome-deficient cells have increased rates of lanosterol biosynthesis compared to wild-type cells. These data are summarized in Table 1 and support the earlier observation that the enzymatic activities of mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isopentenyl diphosphate isomerase, and FPP synthase were significantly reduced in liver tissue from patients with peroxisome-deficient diseases (
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These data are also in agreement with previous studies in peroxisome-deficient human fibroblast cell lines, which also demonstrated impaired rates of cholesterol biosynthesis (
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Literature Cited |
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