Differential binding of proteins to peroxisomes in rat hepatoma cells: unique association of enzymes involved in isoprenoid metabolism.
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
SD
Department of Biochemistry and Molecular Biology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
Mehan
R S
RS
Tansey
T R
TR
Chen
H T
HT
Goping
G
G
Goldberg
I
I
Shechter
I
I
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002463:Cell Membrane Permeability; D006224:Cricetinae; D019084:Fluorescent Antibody Technique, Indirect; D051228:Geranyltranstransferase; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008830:Microbodies / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D012717:Sesquiterpenes; D014407:Tumor Cells, Cultured
D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D006903:Hydroxymethylglutaryl CoA Reductases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase