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First published online January 16, 2004
doi: 10.1242/10.1242/jcs.00910

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Mevalonate kinase is a cytosolic enzyme in humans

¹ Laboratory Genetic Metabolic Diseases, Department of Pediatrics, Emma Children's Hospital, Academic Medical Center, Amsterdam
² Department of Anatomy, Embryology, Histology & Medical Physics, University of Gent, Belgium

View larger version (48K): [in a new window]   Fig. 1. Subcellular fractions of human fibroblasts derived from a control subject (A) or a ZS patient (B) were obtained by Nycodenz equilibrium density gradient centrifugation as described in Materials and Methods. Fractions were analysed for the cytosolic marker PGI (black bars), the peroxisomal marker CAT (grey bars) and MK (open bars). Relative activities were expressed as a percentage of total gradient activity present in each fraction. The pattern of distribution of MK activity and MK protein as determined by immunoblot analysis with an affinity purified antibody raised against human MK was similar to the pattern of PGI activity. Human fibroblasts derived from a control subject (C) or a ZS patient (D) were incubated with increasing concentrations of digitonin as described in Materials and Methods. Supernatant (open symbols) and pellet (closed symbols) fractions were analysed for the activities of the cytosolic marker PGI (square), the peroxisomal marker CAT (triangle) and MK (circle). Relative activities were expressed as a percentage of total activity (supernatant + pellet) present in each fraction. The pattern of latency of MK activity and MK protein as determined by immunoblot analysis with an affinity purified antibody raised against human MK were similar to the pattern of PGI activity.

View larger version (53K): [in a new window]   Fig. 2. Human fibroblasts derived from a control subject (A-D) or a ZS patient (E-H) were labelled with antibodies as described in Materials and Methods. Cells were double labelled using antibodies directed against MK (A,E) and the peroxisomal marker CAT (B,F) or with antibodies directed against MK (C,G) and the cytosolic marker MMP7 (D,H). MK shows the same pattern as the cytosolic MMP7 in both cell lines. MK shows colocalisation with CAT in the ZS fibroblasts in which CAT is localised in the cytosol but no colocalisation is observed between MK and the peroxisomal CAT in control fibroblasts.

View larger version (50K): [in a new window]   Fig. 3. Subcellular fractions of human fibroblasts derived from an FHC patient (A), HEK (B) cells or CV1 cells (C) overexpressing full-length human MK were obtained by Nycodenz equilibrium density gradient centrifugation as described in Materials and Methods. Fractions were analysed for the cytosolic marker PGI (black bars), the peroxisomal marker CAT (grey bars) and MK (open bars). Relative activities were expressed as a percentage of total gradient activity present in each fraction. The patterns of distribution of MK activity and MK protein as determined by immunoblot analysis with an affinity purified antibody raised against human MK were similar to the pattern of PGI activity. Human fibroblasts derived from an FHC patient (D), HEK cells (E) or CV1 cells (F) overexpressing full-length human MK were incubated with increasing concentrations of digitonin as described in Materials and Methods. Supernatant (open symbols) and pellet (closed symbols) fractions were analysed for the activities of the cytosolic marker PGI (square), the peroxisomal marker CAT (triangle) and MK (circle). Relative activities were expressed as a percentage of total activity (supernatant + pellet) present in each fraction. The patterns of latency of MK activity and MK protein as determined by immunoblot analysis with an affinity purified antibody raised against human MK were similar to the pattern of PGI activity.

View larger version (86K): [in a new window]   Fig. 4. Human fibroblasts derived from an FHC patient (A-D), HEK cells (E-H) or CV1 cells (I-L) overexpressing full-length human MK were labelled with antibodies as described in Materials and Methods. Cells were double labelled using antibodies directed against MK (A,E,I) and the peroxisomal marker CAT (B,F,J) or with antibodies directed against MK (C,G,K) and the cytosolic marker MMP7 (D,H,L). The diffuse distribution pattern of MK differs from the punctate pattern of CAT, but MK shows the same pattern as the cytosolic MMP7 in all cell lines.

View larger version (30K): [in a new window]   Fig. 5. CV1 cells overexpressing full-length human MK with an artificial PTS1 signal (SKL) were labelled with antibodies as described in Materials and Methods. Cells were double labelled using antibodies directed against MK (A) and a peroxisomal marker CAT (B). The overlay image of both signals (C) shows a clear colocalisation of MK and CAT in the majority of peroxisomes. In addition, cytosolic labelling of MK can be observed (upper left corner).

View larger version (191K): [in a new window]   Fig. 6. Electron microscopy (A,B) and light microscopy (C,D) of human control liver. (A) Ultrathin Unicryl sections of human liver were immunostained with the affinity purified antibodies against MK. The peroxisomes (P) remain unlabelled. (B) Ultrathin Unicryl sections immunostained with antibodies against against AGT reveals a clear localisation in the peroxisomal matrix. Bar, 500 nm; M, mitochondria. (C) Cryostat sections immunostained with affinity purified antibodies against MK (silver enhancement of colloidal gold) reveals a diffuse reaction in the cytosol of the hepatocytes. For comparison, immunostaining with antibodies against AGT reveals a distinct granular pattern, reflecting a peroxisomal localisation of AGT (D).

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