Cloning, Expression, and Cellular Localization of a Human Prenylcysteine Lyase

Abstract

Prenylated proteins contain either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus. These proteins constitute up to 2% of total cellular protein in eukaryotic cells. The degradation of prenylated proteins raises a metabolic challenge to the cell, because the thioether bond of the modified cysteine is quite stable. We recently identified and isolated an enzyme termed prenylcysteine lyase that cleaves the prenylcysteine to free cysteine and an isoprenoid product (Zhang, L., Tschantz, W. R., and Casey, P. J. (1997) J. Biol. Chem. 272, 23354 -23359). To facilitate the molecular characterization of this enzyme, its cloning was undertaken. Overlapping cDNA clones encoding the complete coding sequence of this enzyme were obtained from a human cDNA library. The open reading frame of the gene encoding prenylcysteine lyase is 1515 base pairs and has a nearly ubiquitous expression pattern with a message size of 6 kilobase pairs. Recombinant prenylcysteine lyase was produced in a baculovirus-Sf9 expression system. Analysis of both the recombinant and native enzyme revealed that the enzyme is glycosylated and contains a signal peptide that is cleaved during processing. Additionally, the subcellular localization of this enzyme was determined to be lysosomal. These findings strengthen the notion that prenylcysteine lyase plays an important role in the final step in the degradation of prenylated proteins and will allow further physiological and biochemical characterization of this enzyme.

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles

“…Furthermore, Vps45, a protein that traffi cs between the TGN and endosomes ( 30 ), was poorly resolved from the late endosomal and TGN marker proteins in fractions 9 and 10. We also examined the distribution of the lysosomal marker prenylcysteine lyase ( 31 ) and found that while it was mainly confi ned to the dense fractions 11 and 12, there was also a substantial amount in the TGN and late endosomal fractions. From these data, we infer that widely employed sucrose gradient centrifugation protocols do not clearly separate the TGN from late endosomal membranes or lysosomes.…”

Prenylcysteine Lyase Deficiency in Mice Results in the Accumulation of Farnesylcysteine and Geranylgeranylcysteine in Brain and Liver

“…The stability of the modification and the abundance of isoprenylated proteins in cells suggested the existence of an enzymatic mechanism for degrading and disposing of prenylcysteine residues. Such an enzyme, termed prenylcysteine lyase (Pcly), 2 was recently identified (7)(8)(9). Pcly is a 505-amino acid flavin adenine dinucleotidedependent thioether oxidase and is located within lysosomes (8,9).…”

“…Such an enzyme, termed prenylcysteine lyase (Pcly), 2 was recently identified (7)(8)(9). Pcly is a 505-amino acid flavin adenine dinucleotidedependent thioether oxidase and is located within lysosomes (8,9). Pcly recognizes both farnesylcysteine and geranylgeranylcysteine and their methyl esters with high affinity and cleaves the thioether bond to yield free cysteine (or cysteine methyl ester) and the aldehyde of the isoprenoid lipid (7).…”

Thematic review series: Lipid Posttranslational Modifications. Lysosomal metabolism of lipid-modified proteins

“…A human cDNA clone encoding the enzyme was obtained based upon the amino acid sequence of the purified bovine enzyme (34). Northern blot analysis showed an z6 kb major transcript, which was ubiquitously expressed, with highest expression in liver.…”