Human mevalonate pyrophosphate decarboxylase is localized in the cytosol.

In the past decade several reports have claimed that peroxisomes play a critical role in the isoprenoid/cholesterol biosynthetic pathway based on the finding of a predominant peroxisomal localization of several of the enzymes involved. Other reports, however, do not support the peroxisomal localization of these enzymes. In this study we have studied the subcellular localization of one of the enzymes, human mevalonate pyrophosphate decarboxylase, by conventional subcellular fractionation and digitonin permeabilization studies, immunofluorescence microscopy, and immunoelectron microscopy. We found a cytosolic localization for both endogenous human mevalonate pyrophosphate decarboxylase (in human fibroblasts, liver, CV1 and HEK293 cells) and overexpressed mevalonate pyrophosphate decarboxylase (in human fibroblasts, HEK293 and CV1 cells) but no indication for a peroxisomal localization. Our results do not support a central role of peroxisomes in the isoprenoid/cholesterol biosynthetic pathway.

Phosphomevalonate kinase is a cytosolic protein in humans.

In the past decade, a predominant peroxisomal localization has been reported for several enzymes functioning in the presqualene segment of the cholesterol/isoprenoid biosynthesis pathway. More recently, however, conflicting results have been reported raising doubts about the postulated role of peroxisomes in isoprenoid biosynthesis, at least in humans. In this study, we have determined the subcellular localization of human phosphomevalonate kinase using a variety of biochemical and microscopic techniques, including conventional subcellular fractionation studies, digitonin permeabilization studies, immunofluorescence, and immunoelectron microscopy. We found an exclusive cytosolic localization of both endogenously expressed human phosphomevalonate kinase (in human fibroblasts, human liver, and HEK293 cells) and overexpressed human phosphomevalonate kinase (in human fibroblasts, HEK293 cells, and CV1 cells). No indication of a peroxisomal localization was obtained. Our results do not support a central role of peroxisomes in isoprenoid biosynthesis.

Mevalonate kinase is a cytosolic enzyme in humans.

In the past decade several reports have appeared which suggest that peroxisomes play a central role in isoprenoid/cholesterol biosynthesis. These suggestions were based primarily on the reported finding of several of the enzymes of the presqualene segment of the biosynthetic pathway in peroxisomes. More recently, however, conflicting results have been reported raising doubt about the postulated role of peroxisomes in isoprenoid biosynthesis, at least in humans. In this study we have studied the subcellular localisation of human mevalonate kinase (MK) using a variety of biochemical and microscopical techniques. These include conventional subcellular fractionation studies, digitonin permeabilisation studies, immunofluorescence microscopy and immunocytochemistry. We exclusively found a cytosolic localisation of both endogenous human MK (human fibroblasts, liver and HEK293 cells) and overexpressed human MK (human fibroblasts, HEK293 cells and CV1 cells). No indication of a peroxisomal localisation was obtained. Our results do not support a central role for peroxisomes in isoprenoid biosynthesis.

Cholesterol biosynthesis is not defective in peroxisome biogenesis defective fibroblasts.

To evaluate the presumed peroxisomal involvement in cholesterol/isoprenoid biosynthesis, we determined the protein levels and activities of five different enzymes of the presqualene segment of the cholesterol/isoprenoid biosynthetic pathway in primary skin fibroblasts of selected patients with a peroxisomal biogenesis disorder (PBD). These five enzymes all have been reported to be partly or exclusively peroxisomal and include HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, and isopentenyl pyrophosphate isomerase. To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, have differential effects on the activity of the cholesterol biosynthetic enzymes and on de novo cholesterol biosynthesis, we chose fibroblasts of patients with defined defects in one of four different PEX genes leading to Zellweger syndrome (PEX1, PEX5, PEX16 or PEX19). We found that all enzymes measured are at least as active in the peroxisome-deficient cells cultured in cholesterol-depleted medium as in identically cultured control cells. This indicates that if these presumed peroxisomal proteins are mislocalized to the cytosol they do not loose their activity, nor get degraded unlike most other authentic peroxisomal proteins. We also measured de novo cholesterol synthesis from radio-labeled acetate in all cell lines and found similar or even elevated rates for the PBD cells when compared to controls. Our results imply that functional peroxisomes are not a prerequisite for the functioning of enzymes involved in cholesterol/isoprenoid biosynthesis and as such raise doubts about the true involvement of peroxisomes therein.

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis.

To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger syndrome. We found that all enzymes measured, including putative peroxisomal enzymes, are at least as active in the peroxisome-deficient Zellweger mice as in control mice, indicating that mislocalization of enzymes to the cytosol does not lead to decreased activity or degradation. Prompted by these results, we re-examined this aspect in human subjects by specific enzyme activity measurements and immunoblotting with highly specific antisera. Our results show that the previously reported deficiencies of mevalonate kinase and phosphomevalonate kinase activity in livers from human Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol. Our data provide an explanation for the conflicting findings in the literature and show that great care should be taken in the interpretation of data obtained in postmortem material.