Complexity of Trypanosomatid Endocytosis Pathways Revealed by Rab4 and Rab5 Isoforms in Trypanosoma brucei

Abstract

Small G proteins of the Rab family are responsible for vesicle fusion and control flux during intracellular transport. Rab5 is important in endosome maturation and Rab4 in recycling of endocytic material. Three Rab5 isoforms identified so far in mammals and three in the yeast genome suggest that conservation of multiple Rab5 isoforms is required for sophisticated regulation of endocytosis. Trypanosoma brucei homologues of Rab5 and Rab4 (TbRab5A and TbRab4) have been identified. Here we report cloning of a second Rab5 homologue, TbRab5Bp. The TbRAB5A and -5B genes are not linked in the genome, and phylogenetic reconstruction indicates that multiple Rab5 isoforms in yeast, mammals, and trypanosomes evolved independently. Northern blots demonstrate that TbRab5A, -5B, and TbRab4 messages are expressed in bloodstream form (BSF) and procyclic forms of the parasite even though endocytosis is not very active in the latter form. mRNA levels of TbRab5A and -4 are constitutive. Multiple-sized TbRab5B messages at very low abundance are detected, with greater expression in BSF. Also, the TbRab5B mRNA has a large 3-untranslated region suggestive of potentially complex regulation, and therefore TbRab5Bp may be an important regulator of differential endocytosis levels between BSF and procyclic stage parasites. Affinity purified antibodies raised to C-terminal peptide sequences of all three TbRab proteins recognized small vesicular cytoplasmic structures, which for TbRab5Ap and -5Bp are predominantly near the flagellar pocket. TbRab5Bp colocalizes with invariant surface glycoprotein 100 (ISG 100 ), a protein entering the endocytotic pathway in BSF parasites, whereas in procyclic cells populations of vesicles stained with both TbRab5Ap and -5Bp substantially overlap; TbRab5 proteins are therefore components of the endocytotic pathway. TbRab4p localizes to vesicular structures throughout the cytoplasm, with some overlap with TbRab5Bp, but the majority occupying a different compartment to the TbRab5s. Therefore the trypanosome endosomal system has been functionally dissected for the first time; these reagents provide a unique opportunity for manipulation of the protozoan endosomal system to further our understanding of drug uptake mechanisms and virulence.

Rab5-associated Vacuoles Play a Unique Role in Phagocytosis of the Enteric Protozoan Parasite Entamoeba histolytica

“…This is in good contrast to the yeast and mammalian counterparts, which are virtually interchangeable; Ypt51p expressed in a mammalian cell was properly localized to endosomes and accelerated endocytosis (6). Fifth, compared with other organisms including mammals, plants, yeasts, and a parasitic protist Trypanosoma brucei, which have been shown to possess 2-5 Rab5 isotypes with distinct tissue and organelle distribution or developmental stage-specific expression (46,(57)(58)(59), E. histolytica possesses only a single Rab5 gene based on our thorough search of the genome data base (data not shown). Altogether, EhRab5 represents a unique Rab5 showing diverse localization and functions.…”

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways

“…However, the location of T. brucei Rab28 was consistent with association with one or more of these compartments, especially the endosomes and lysosome (Field et al, 1998;Jeffries et al, 2001;Gabernet-Castello et al, 2009;Leung et al, 2008;Alexander et al, 2002).…”

Phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution

“…Rab 5 paralogues in yeast also appear to possess distinct functions, although some redundancy likely exists (40), and similarly nonredundant functionality has been described for mammalian Rab 5 isoforms (41). Previous phylogenetic analyses of Rab 5 paralogues from these three taxa suggested that the last common ancestor of this gene in trypanosomes, yeast, and humans are derived from a single Rab5 gene that was then duplicated in these lineages (42). However, the analysis did not encompass diverse eukaryotic taxa or use methods accounting for rate variation, an important factor that can cause artifacts in phylogenetic reconstruction (31).…”