Characterisation of protein isoprenylation in procyclic form Trypanosoma brucei

Protein modification by isoprenylation is essential in mammals and other eukaryotes, but has not been demonstrated in the parasitic protozoa of the order kinetoplastida. A key regulatory enzyme of the mevalonate 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 the presence of compactin, an efficient inhibitor of HMG-R, followed by one-dimensional gel electrophoresis, we demonstrate that protein isoprenylation indeed takes place in this organism and at least 14 polypeptides bear the modification. Further characterization of labelled isoprenyl groups by methyl iodide cleavage and high pressure liquid chromatography identified both the farnesyl and geranylgeranyl moieties found covalently attached to proteins in other eukaryotes. The latter moiety was more abundant, as found in mammalian systems. Prolonged incubation with compactin grossly affected cell morphology and altered a number of subcellular structures as seen by electron microscopy. High concentrations of compactin were toxic, whilst lower concentrations were cytostatic. The primary morphological lesion is distinct from that of synvinolin, another inhibitor of HMG-R. The morphological changes correlated with a complete inhibition of HMG-R activity by compactin. Surprisingly there was a complete lack of HMG-R activity in procyclic cells grown for 1 or several days in 100 microM compactin, suggesting that degradation of the enzyme had occurred and compensatory upregulation mechanisms could not be successfully exploited by the parasite to overcome HMG-R inhibition. Subsequent alterations to the overall cell shape are seen after 3 days of compactin exposure. Overall these data indicate that T. brucei has an essential protein isoprenylation pathway that is conserved with the higher eukaryotes. Additionally, products of the MVA pathway are implicated in maintenance of cell architecture.