Differential binding of proteins to peroxisomes in rat hepatoma cells: unique association of enzymes involved in isoprenoid metabolism

Sita D Gupta,Ryan S Mehan,Terese R Tansey,Hua-Tang Chen,Gertrud Goping,Israel Goldberg,Ishaiahu Shechter

doi:10.1016/s0022-2275(20)33403-9

Sita D Gupta, Ryan S Mehan + Show 5 more

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https://doi.org/10.1016/s0022-2275(20)33403-9

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Abstract

Farnesyl diphosphate synthase (FPPS: EC2.5.1.10), a key enzyme in isoprenoid metabolic pathways, catalyzes the synthesis of farnesyl diphosphate (FPP) an intermediate in the biosynthesis of both sterol and non-sterol isoprenoid end products. The localization of FPPS to peroxisomes has been reported (Krisans, S. K., J. Ericsson, P. A. Edwards, and G. A. Keller. 1994. J. Biol. Chem. 269: 14165–14169). Using indirect immunofluorescence and immunoelectron microscopic techniques we show here that FPPS is localized predominantly in the peroxisomes of rat hepatoma H35 cells. However, the partial release of 60–70% of cellular FPPS activity is observed by selective permeabilization of these cells with digitonin. Under these conditions, lactate dehydrogenase, a cytosolic enzyme, is completely released whereas catalase, a known peroxisomal enzyme, is fully retained. Digitonin treatment of H35 cells differentially affects the release of other peroxisomal enzymes involved in isoprenoid metabolism. For instance, mevalonate kinase and phosphomevalonate kinase are almost totally released (95% and 91%, respectively), whereas 3-hydroxy-3-methylglutaryl-CoA reductase is fully retained. Indirect immunoflourescence studies indicate that FPPS is localized in peroxisomes of Chinese hamster ovary (CHO)-K1 cells but is dispersed in the cytosol of ZR-82 cells, a mutant that lacks peroxisomes. Unlike in H35 cells, FPPS is completely released upon digitonin permeabilization of CHO-K1 and ZR-82 cells. In contrast, under the same permeabilization conditions, catalase is fully retained in CHO-K1 cells but completely released from ZR-82 cells.▪ These studies indicate that FPPS and other enzymes in the isoprenoid biosynthetic pathways, involved in the formation of FPP, are differentially associated with peroxisomes and may easily diffuse to the cytosol. Based on these observations, the significance and a possible regulatory model in the formation of isoprenoid end-products are discussed.—Gupta, S. D., R. S. Mehan, T. R. Tansey, H-T. Chen, G. Goping, I. Goldberg, and I. Shechter. Differential binding of proteins to peroxisomes in rat hepatoma cells: unique association of enzymes involved in isoprenoid metabolism. J. Lipid Res. 1999. 40: 1572–1584.

Highlights

Farnesyl diphosphate synthase (FPPS: EC2.5.1.10), a key enzyme in isoprenoid metabolic pathways, catalyzes the synthesis of farnesyl diphosphate (FPP) an intermediate in the biosynthesis of both sterol and non-sterol isoprenoid end products
Preliminary studies involving subcellular fractionation of these cells resulted in the release of the majority of the FPPS activity into the cytosolic supernatant similar to the results shown by Krisans et al [9]
In both cell lines the major amount of FPPS is released upon permeabilization, in agreement with the results shown in Enzyme lactate dehydrogenase (LDH) Catalase FPPS

Summary

Introduction

Farnesyl diphosphate synthase (FPPS: EC2.5.1.10), a key enzyme in isoprenoid metabolic pathways, catalyzes the synthesis of farnesyl diphosphate (FPP) an intermediate in the biosynthesis of both sterol and non-sterol isoprenoid end products. Under the same permeabilization conditions, catalase is fully retained in CHO-K1 cells but completely released from ZR-82 cells These studies indicate that FPPS and other enzymes in the isoprenoid biosynthetic pathways, involved in the formation of FPP, are differentially associated with peroxisomes and may diffuse to the cytosol. Studies by several investigators have shown that the enzymes mevalonate kinase [5, 6], phosphomevalonate kinase [7], diphosphomevalonate decarboxylase [7], and isopentenyl diphosphate dimethylallyl diphosphate (IPP) isomerase [8] which produce intermediates for the synthesis of farnesyl diphosphate (FPP), and FPP synthase (FPPS) [9], are localized in peroxisomes [1] Based on these findings it was proposed that the conversion of mevalonate to FPP may occur exclusively in peroxisomes (5 –9).

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