Abstract
Endocytosis is a crucial process in eukaryotic cells. The GTPases Rab 5, 21 and 22 that mediate endocytosis are ancient eukaryotic features and all available evidence suggests retained conserved function. In animals and fungi, these GTPases are regulated in part by proteins possessing Vps9 domains. However, the diversity, evolution and functions of Vps9 proteins beyond animals or fungi are poorly explored. Here we report a comprehensive analysis of the Vps9 family of GTPase regulators, combining molecular evolutionary data with functional characterization in the non‐opisthokont model organism Trypanosoma brucei. At least 3 subfamilies, Alsin, Varp and Rabex5 + GAPVD1, are found across eukaryotes, suggesting that all are ancient features of regulation of endocytic Rab protein function. There are examples of lineage‐specific Vps9 subfamily member expansions and novel domain combinations, suggesting diversity in precise regulatory mechanisms between individual lineages. Characterization of the Rabex5 + GAPVD1 and Alsin orthologues in T. brucei demonstrates that both proteins are involved in endocytosis, and that simultaneous knockdown prevents membrane recruitment of Rab5 and Rab21, indicating conservation of function. These data demonstrate that, for the Vps9‐domain family at least, modulation of Rab function is mediated by evolutionarily conserved protein‐protein interactions.
Highlights
During endocytosis, cells take up and deliver extracellular and surface material to early endosomes
Characterization of the Rabex5 + GAPVD1 and Alsin orthologues in T. brucei demonstrates that both proteins are involved in endocytosis, and that simultaneous knockdown prevents membrane recruitment of Rab[5] and Rab[21], indicating conservation of function
As excavate subclade phylogenies were poorly resolved (Figure S1K), in order to classify the T. brucei Vps[9] proteins, we constructed trees with only the trypanosome and metazoa backbone sequences. We found that these sequences group with Alsin (Tb927.3.2430, designated here TbAlsin) and Rabex5 + GAPVD1 (Tb927.10.10020, designated here TbRabex5) (Figure 3)
Summary
Cells take up and deliver extracellular and surface material to early endosomes. While there is a canonical pan-eukaryotic complement of Rabs and Rab effectors, there have been numerous lineage-specific expansions and contractions, likely as part of the process of adaptation to specific environments.[1,14] In line with this, though Rab[5] is ancient and found across eukaryotes, it has undergone independent gene duplication events in many lineages, suggesting ongoing specialization of the early endocytic system.[14,15] Like Rab[5], multiple Vps[9] domaincontaining GEFs are present in mammals: Vps9/Rabex[5], VPS9ankyrin-repeat protein (Varp), Ras and Rab Interactors (RIN) 1-3, Alsin, GAPVD1 and Vps[9] domain-containing protein 1 (Vps9DCP1).[16] Each of these proteins is involved in interactions with Rab[5], or its close relatives, and plays a role in endocytosis in animal or yeast cells. In order to determine how Vps[9] family proteins may function in organisms outside of the Opisthokonta (animals and fungi), we performed localization and knockdown experiments of the 2 Vps[9] paralogues in the excavate parasite Trypanosoma brucei, a divergent organism with a comparatively well-characterized endosomal system
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
