CHARACTERIZATION OF PROTEIN ISOPRENYLATION IN PROCYCLIC FORM TRYPANOSOMA-BRUCEI

Protein modification by isoprenylation is essential in mammals and oth er eukaryotes, but has not been demonstrated in the parasitic protozoa of the order kinetoplastida. A key regulatory enzyme of the mevalonat e pathway, hydroxymethylglutaryl-coenzyme A reductase (HMG-R), and end products of the path, including dolichols, are present in Trypanosoma brucei. By metabolical labelling of procyclic form trypanosomes in th e presence of compactin, an efficient inhibitor of HMG-R, followed by one-dimensional gel electrophoresis, we demonstrate that protein isopr enylation indeed takes place in this organism and at least 14 polypept ides bear the modification. Further characterization of labelled isopr enyl groups by methyl iodide cleavage and high pressure liquid chromat ography identified both the farnesyl and geranylgeranyl moieties found covalently attached to proteins in other eukaryotes. The latter moiet y was more abundant, as found in mammalian systems. Prolonged incubati on with compactin grossly affected cell morphology and altered a numbe r of subcellular structures as seen by electron microscopy. High conce ntrations of compactin were toxic, whilst lower concentrations were cy tostatic. The primary morphological lesion is distinct from that of sy nvinolin, another inhibitor of HMG-R. The morphological changes correl ated with a complete inhibition of HMG-R activity by compactin. Surpri singly there was a complete lack of HMG-R activity in procyclic cells grown for 1 or several days in 100 mu M compactin, suggesting that deg radation of the enzyme had occurred and compensatory upregulation mech anisms could not be successfully exploited by the parasite to overcome HMG-R inhibition. Subsequent alterations to the overall cell shape ar e seen after 3 days of compactin exposure. Overall these data indicate that T. brucei has an essential protein isoprenylation pathway that i s conserved with the higher eukaryotes. Additionally, products of the MVA pathway are implicated in maintenance of cell architecture.