Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering.

Center for Computational Sciences University of Tsukuba Tsukuba Japan

Department of Biochemistry and Robert Cedergren Centre for Bioinformatics and Genomics Université de Montréal Montreal Canada

Department of Biology and Ecology and Institute of Environmental Technologies Faculty of Science University of Ostrava Ostrava Czech Republic

Department of Botany University of British Columbia Vancouver Canada

Department of Genetics Evolution and Environment University College London London United Kingdom

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

Institute for Biological Sciences University of Tsukuba Tsukuba Japan

Laboratory of Microbiology Papanin Institute for Biology of Inland Waters Russian Academy of Sciences Borok Russia

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