3-Polyprenyl-4-hydroxybenzoate Synthesis in the Inner Membrane of Mitochondria from p-Hydroxybenzoate and Isopentenylpyrophosphate A DEMONSTRATION OF ISOPRENOID SYNTHESIS IN RAT LIVER MITOCHONDRIA

Abstract The first intermediate in the conversion of p-hydroxybenzoate (PHB) to ubiquinone is a 3-polyprenyl derivative of PHB. Winrow and Rudney ((1969) Biochem. Biophys. Res. Commun. 37, 833) noted that in rat tissue minces and homogenates the synthesis of 3-polyprenyl-4-hydroxybenzoate (PPHB) required supplementation with an extract of Micrococcus lysodeikticus and isopentenylpyrophosphate. Under new conditions adopted by us the requirement for the M. lysodeikticus extract can be dispensed with and the synthesis of polyprenyl-p-hydroxybenzoate can be obtained solely from p-hydroxybenzoate, mevalonate, and ATP. Pulse chase experiments with labeled precursors in rat liver minces show a precursor-product relationship of PPHB and ubiquinone. It was found that PPHB was located primarily in the inner membrane of liver mitochondria. Mitochondrial preparations to which PHB, ATP, and mevalonate were added could not synthesize PPHB unless a supernatant fraction obtained by centrifugation of rat liver homogenates at 48,000 x g was added. The requirement for the supernatant fraction, ATP, and mevalonate could be dispensed with only if isopentenylpyrophosphate were added. This implied that mitochondria contain a system for synthesizing polyprenylpyrophosphate derivatives. This concept was unequivocally verified by demonstration of the presence of a sequence of key enzymes involved in polyprenyl synthesis, i.e. isopentenylpyrophosphate isomerase and diprenyltransferase, in the inner membrane of mitochondria. The nature of the PPHB formed in rat liver was investigated and it was found that in keeping with the known distribution of ubiquinone prenyl analogues UQ-9 and UQ-10 in rat liver the same proportion of 3-decaprenyl-4-hydroxybenzoate and 3-nonaprenyl-4-hydroxybenzoate could be formed. Guinea pig liver which is known to contain only UQ-10 was also shown to form only 3-decaprenyl-4-hydroxybenzoic acid. The enzyme alkylating PHB, i.e. PHB: polyprenyltransferase, has been shown to be unspecific with regard to the length of the polyprenyl side chain which it can transfer o PHB. The predominance of any prenyl analogue of UQ in a tissue depends upon the specificity of the system which determines the length of the polyprenylpyrophosphate chains synthesized in mitochondria. Conditions for the optimal synthesis of PPHB from PHB and isopentenylpyrophosphate in mitochondria are described. The effect of CoA-SH was investigated on PPHB formation and was found to be either nonexistent or stimulatory depending on the medium in which the synthesis was carried out. In the latter case CoA-SH increased the amount of shorter chain prenyl analogues of PHB. The results, providing the first demonstration that a system of enzymes for synthesizing polyprenylpyrophosphate chains exists in the inner mitochondrial membrane, are of great biological significance in view of the increasing role that these compounds play in the transfer of sugars to glycoproteins. Several other possible roles for polyprenylpyrophosphate compounds in mitochondria are discussed.