| Summary: | In eukaryotic cells, the mevalonate pathway of isoprenoid biosynthesis provides the cell with essential precursors for several cellular processes. For example, one product, FDP (farnesyl diphosphate) is a fundamental precursor for sterol biosynthesis and it is also used for protein prenylation. In the slime mould Dictyostelium discoideum some of the mevalonate pathway enzymes possess a peroxisomal targeting signal. This suggested that part of the mevalonate pathway may take place in the peroxisomes. In this study, the intracellular locations of the mevalonate pathway enzymes were investigated by transforming D. discoideum amoebae to express each enzyme as a fusion protein with either GFP (green fluorescent protein) or mRFP (monomeric red fluorescent protein). It was found that three of the mevalonate pathway enzymes are peroxisomal: 3-hydroxy-3-methylglutaryl-coenzyme A synthase isozyme B, phosphomevalonate kinase and farnesyl diphosphate synthase. HMG-CoA reductase is associated with the endoplasmic reticulum and the other four enzymes of the mevalonate pathway were most likely to be in the cytosol: HMG-CoA synthase isozyme A, mevalonate kinase, diphosphomevalonate decarboxylase and IDP-isomerase. The intracellular location of the first five enzymes involved in sterol biosynthesis from farnesyl diphosphate was also identified by using the GFP or mRFP tagged enzyme approach. Some of these enzymes possess a strong peroxisomal targeting signal type 1 (PTS1). It was shown that the first four enzymes of the pathway: squalene synthase, squalene epoxidase, oxidosqualene cyclase and cycloartenol -C-24-methyltransferase are peroxisomal whereas the two isozymes of the fifth enzyme, methylsterol monooxygenase, are associated with the endoplasmic reticulum. It was also demonstrated that the first four enzymes on the sterol biosynthesis pathway are strongly associated with the peroxisomal membrane. However, the putative PTS1 present at the C-terminus of squalene synthase, oxidosqualene cyclase and cycloartenol-C-24-methyltransferase would imply that each of these enzymes should be a peroxisomal matrix protein. We therefore investigated whether the putative PTS1s are involved in directing these three enzymes into the peroxisomes. It was found that squalene synthase was largely peroxisomal even when its PTS1 was absent but the PTS1 in oxidosqualene synthase and cycloartenol-C-24-methyltransferase was essential for entry of these enzymes into peroxisomes. It appears that the sterol biosynthesis in D. discoideum is unusual since the enzymes squalene synthase, squalene epoxidase, oxidosqualene cyclase and cycloartenol-C-24-methyltransferase are accumulated in the peroxisomes whereas in all other organisms studied they are in the endoplasmic reticulum.
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