Myristoyl-CoA protein:NMT (N-myristoyl transferase) catalyses the N-myristoylation of cellular proteins with a range of functions and is essential for viability in the protozoan parasites, Leishmania major and Trypanosoma brucei. In our investigations to define the essential downstream targets of NMT, we have focused on the ARF (ADP-ribosylation factor) family of proteins, as growth arrest in Saccharomyces cerevisiae mutants with reduced NMT activity correlates with decreased modification of members of this group of proteins. We have identified nine ARF/ARLs (where ARL stands for ARF-like) encoded in the T. brucei and T. cruzi genomes and ten in L. major. The T. brucei ARL1 protein is expressed only in the mammalian bloodstream form of the parasite, in which it is localized to the Golgi apparatus. RNAi (RNA interference) has been used to demonstrate that ARL1 is essential for viability in these infective cells. Before cell death, depletion of ARL1 protein results in disintegration of the Golgi structure and a delay in exocytosis of the abundant GPI (glycosylphosphatidylinositol)-anchored VSG (variant surface glycoprotein) to the parasite surface.
Subspecies of Trypanosoma brucei are the causative agents of African trypanosomiasis or sleeping sickness, a disease that threatens over 60 million people in sub-Saharan Africa, and the major veterinary infection, Nagana, in livestock. Infections with these parasites represent a huge economic and social burden in endemic regions (http://www.who.int/tdr/diseases/tryp). T. brucei cycles between two extracellular lifecycle stages, residing either in the mammalian host bloodstream or as a procyclic form in the midgut of the tsetse fly (Glossina) vector. Research on T. brucei, both as a disease agent and as a model lower eukaryote organism, has recently been accelerated by completion of the genome project for T. brucei brucei (www.genedb.org/genedb/tryp) and the development of a robust tetracycline-inducible RNAi (RNA interference) system . This combination provides valuable resources for the study of parasite-specific cellular processes and the development of novel therapeutic agents.
N-myristoylation and ARFs (ADP-ribosylation factors)
The enzyme NMT (N-myristoyl transferase) catalyses the co-translational attachment of the fatty acid myristate to target proteins, a process which is essential for viability in many pathogenic eukaryotic species, including Candida albicans and the kinetoplastid parasites, Leishmania major and T. brucei . There has been considerable interest in the design of specific NMT inhibitors for use as anti-fungal agents, an approach that may have potential for the development of new anti-parasitic drugs . Identifying the essential downstream substrates of NMT that contribute to the lethal phenotype observed in the absence of enzyme activity will aid in elucidation of the mechanisms of parasite killing and could provide additional targets for intervention.
The most extensively studied group of N-myristoylated proteins are the ARFs, a highly conserved family of small GTPases with roles in membrane dynamics and trafficking . The related ARL (ARF-like) proteins demonstrate a wider repertoire of localization and function in the cell, ranging from the regulation of tubulin folding (ARL2)  to the stimulation of cholesterol secretion (ARL7) . In contrast, ARL1 is an N-myristoylated trans-Golgi protein involved in endosome to Golgi traffic, protein sorting and the maintenance of Golgi integrity .
T. brucei ARL1 is essential in bloodstream parasites
There are at least nine members of the ARF/ARL protein family in T. brucei . We recently reported the characterization of T. brucei ARL1, the only known example of this protein to date to be differentially regulated. TbARL1 is expressed in the host bloodstream stage of the parasite but not in the vector procyclic stage . Disruption of ARL1 expression in T. brucei bloodstream parasites by RNAi results in cell death, demonstrating that ARL1 protein is essential for viability. ARL1 is also essential for development in Drosophila  and expression of a dominant-negative protein causes Golgi disintegration in human cells . However, it is not required for viability in Saccharomyces cerevisiae, with gene deletion resulting in minor defects in protein trafficking .
Before death, ARL1-depleted T. brucei bloodstream cells develop a number of morphological abnormalities, including the accumulation of vesicles and multivesicular bodies and disintegration of the Golgi apparatus (Figure 1). Cells are endocytically active but show a delay in the exocytosis of the highly abundant GPI (glycosylphosphatidylinositol)-anchored protein, VSG (variant surface protein) . As ARL1 is essential for viability and Golgi integrity in bloodstream parasites but undetectable in procyclic cells, this suggests some fundamental differences in Golgi structure and function in the two life-cycle stages of T. brucei.
Electron micrographs of typical
T. brucei bloodstream form of parasites before (A) and after (B) the disruption of ARL1 expression by RNAi (36 h post-induction)
Differences in the requirement for ARL1 during the life cycle?
These observations raise the question of whether the known effectors of ARL1, such as GRIP domain proteins , are recruited by an alternative molecule in procyclic cells or whether they are also absent in this life-cycle stage and are instead replaced by components of an alternative pathway. The reasons why such an arrangement might have evolved are not immediately apparent, although the two parasite stages thrive under contrasting environmental conditions and display distinct differences in morphology, biochemistry and metabolic requirements. The most striking difference between insect and host stages of T. brucei to date is in the rate of cellular protein trafficking, including transport through the flagellar pocket, a deep invagination of the plasma membrane which acts as the sole site of exocytosis and endocytosis in the cell . As extracellular pathogens, bloodstream T. brucei are under constant pressure from the host immune system. The dominant surface protein VSG has a major role in host immune evasion, undergoing antigenic variation to allow persistent infection (reviewed in ). The protein is also rapidly endocytosed, clearing surface-bound immunoglobulins, before recycling back on to the plasma membrane. The entire surface VSG of the parasite has a turnover time of 12 min, more than double the membrane turnover rates reported in mammalian cells . The relevant internal machinery of the cell needs to be channelled towards the maintenance of this vital process. In contrast, protein transport is much slower in the procyclic stage, consistent with a significant down-regulation of proteins functioning in endocytosis, such as clathrin .
Another major difference between the two life-cycle stages is in their metabolic requirements. Bloodstream cells are entirely dependent on glycolysis for their ATP supply , metabolizing glucose into pyruvate in specialized peroxisome-like organelles, the glycosomes. Energy metabolism is considerably more complex in insect-stage cells, characterized by aerobic fermentation of glucose when available, and the additional utilization of lipids and amino acids, particularly proline, which is used by the tsetse fly as its main energy source during flight .
It is feasible that the need to prioritize sorting and trafficking of very different sets of enzymes, receptors, effectors and metabolites in the two life-cycle stages of T. brucei has exerted sufficient evolutionary pressure for the specialization of Golgi function in these cell types. Ongoing research will produce further insights into these mechanisms in T. brucei and related pathogenic organisms.
Localization and Activation of Ras-like GTPases: Focused Meeting held at the Royal Agricultural College, Cirencester, U.K., 21–23 March 2005. Organized and Edited by A. Ridley (Ludwig Institute of Cancer Research, London, U.K.) and M. Seabra (Imperial College London, U.K.).
We thank G. Cross and D. LaCount for parasite strains and the p2T7Ti vector construct. We also thank C. Guerra and other members of the Smith group for helpful discussions. This work was funded by the Wellcome Trust (061343/Z/00/Z).