Regulation of early endosomes across eukaryotes: Evolution and functional homology of Vps9 proteins

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.

Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29

“…VARP, VPS29 and TBC1D5 are all ancient proteins, present in the Last Eukaryotic Common Ancestor 5 , 55 , 56 . However, co-occurrence need not necessarily indicate interaction.…”

“…VARP homologues were identified using HMMer 73 . A hidden Markov model was generated using phylogenetically verified holozoan VARP homologues published in Herman et al 56 , which was used to search predicted proteins from the genomes listed in Supplementary Table 2. All hits were then used as queries in BLASTP searches into the Homo sapiens genome 74 .…”

Allosteric Structural Alterations and Auto-regulation of Rab5 GEF Activity in Rabex5

“…A study of the evolution of the Vps9 domain showed 3 independent instances of acquisition of structurally diverse UBDs: the ZnF in mammalian Rabex5, the CUE domain in yeast, and a UBD in the amebozoan Sexangularia sp. (Herman, Ali et al, 2018). The fact that Vps9 domain containing proteins had such a strong tendency to acquire UBD through independent means suggests a critical role for UBDs in regulating Vps9 activity.…”

Adaptation and Therapeutic Exploitation of the Plasma Membrane of African Trypanosomes

“…Subsequently, the endocytic process involves transitioning through additional compartments that are defined by Rab21 (Tb927.10.1520), Rab28 (Tb927.6.3040), and finally Rab7 (Tb927.9.11000) [ 29 , 30 , 31 ]. Quite what the specific functions of these steps are remains to be elucidated but control does appear to be mediated by the vacuolar protein sorting 9 (Vps9) domain proteins, such as Alsin (Tb927.3.2430) and Rabex5 + GAPVD1 (Tb927.10.10020), a mechanism that is conserved with mammalian cells [ 32 ].…”