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.
Highlights
In eukaryotic organisms, covalent modification by lipids of certain proteins plays an important role in the subcellular localization and biological activities of these proteins [1]
In an ongoing effort to elucidate the metabolic fate of prenylcysteines in cells, we recently identified an enzyme that catalyzes the degradation of prenylcysteines that we have dubbed prenylcysteine lyase (PCLase;1 Ref. 15)
Northern blot analysis showed a nearly ubiquitous expression pattern with the highest level found in the liver. Both sequence analysis and experimental data demonstrate the presence of a cleavable signal peptide in PCLase, and experiments using both the native bovine enzyme and recombinant protein revealed that PCLase is a glycosylated enzyme
Summary
Peptide Sequencing—PCLase was purified from the membrane fraction of bovine brain as described previously [15]. All clones obtained via this procedure were determined by in vitro translation (TNT system; Promega) to be missing their initiating methionine, and 5Ј rapid amplification of cDNA ends (5Ј RACE) was used to obtain the 5Ј end using human brain poly(A)ϩ mRNA (CLONTECH) [21]; a 5Ј RACE product containing the 5Ј end of the cDNA (see “Results”) was obtained and subcloned into a partial cDNA clone from the Gene Trap using a unique Nru site near the 5Ј end of the open reading frame This final vector was termed pCMV-Sport/PCLase and contained the entire open reading frame of PCLase. The sequences obtained from peptides isolated from tryptic digests of purified PCLase, and that from the N-terminal analysis, are shown
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