Extensive molecular tinkering in the evolution of the membrane attachment mode of the Rheb GTPase

Abstract

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.

Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

“…For this purpose, we generated a Rheb mutant that lacks the C-terminal CaaX motif and therefore cannot be farnesylated but instead contains a myristoylation signal at its N-terminus (Figure 5H). This strategy was based on the following logic: 1) myristoylation, like farnesylation, supports only transient membrane interactions; 2) N-terminal myristoylation was previously shown to be an effective substitute for C-terminal farnesylation in H-Ras (Cadwallader et al , 1994); and 3) recent phylogenetic analysis identified Rheb genes in some species that lack C-terminal farnesylation but are predicted to be myristoylated at their N-termini (Zahonova et al , 2018). Interestingly, although a Rheb mutant that is neither farnesylated nor myrisotylated failed to promote mTORC1 signaling in Rheb Depleted cells, myristoylated Rheb stimulated S6K phosphorylation (Figure 5, I and J).…”

Weak Membrane Interactions Allow Rheb to Activate mTORC1 Signaling Without Major Lysosome Enrichment

“…4H). This strategy was based on the following logic: 1) Myristoylation, like farnesylation supports only transient membrane interactions; 2) N-terminal myristoylation was previously shown to be an effective substitute for C-terminal farnesylation in H-Ras (Cadwallader et al, 1994); and 3) Recent phylogenetic analysis identified Rheb genes in some species that lack C-terminal farnesylation but are predicted to be myristoylated at their N-termini (Zahonova et al, 2018). Interestingly, although a Rheb mutant that is neither farnesylated or myrisotylated failed to promote mTORC1 signaling in Rheb Depleted cells, myristoylated Rheb stimulated S6K phosphorylation ( Fig.…”

“…Third, constitutively targeting TSC to lysosomes suppresses mTORC1 signaling (Menon et al, 2014). Fourth, although the Rheb protein in most commonly studied model organisms is targeted to membranes via farnesylation of a C-terminal CaaX motif, a recent phylogenetic analysis of Rheb protein sequences revealed that Rheb homologs in some species lack lipidation and are instead predicted to interact with membranes via an N-terminal FYVE domain (Zahonova et al, 2018). In others, the C-terminal CaaX motif is accompanied by an N-terminal PX domain (Zahonova et al, 2018).…”

“…Fourth, although the Rheb protein in most commonly studied model organisms is targeted to membranes via farnesylation of a C-terminal CaaX motif, a recent phylogenetic analysis of Rheb protein sequences revealed that Rheb homologs in some species lack lipidation and are instead predicted to interact with membranes via an N-terminal FYVE domain (Zahonova et al, 2018). In others, the C-terminal CaaX motif is accompanied by an N-terminal PX domain (Zahonova et al, 2018). Although these bioinformatic predictions remain to be experimentally validated, PX and FYVE domains commonly recruit proteins to endosomal membranes via their ability to bind to phosphatidylinositol-3-phosphate (PI3P) with modest affinity (Burd and Emr, 1998) (Gaullier et al, 1998).…”

Peculiar features of the plastids of the colourless alga Euglena longa and photosynthetic euglenophytes unveiled by transcriptome analyses

“…The sequencing data were obtained from cultures grown at two different light regimes (in the dark and in the light) to improve the coverage of differentially expressed genes and to maximize the chance we detect possible traces of genes encoding the photosynthesis-related machinery. Data extracted from the sequenced E. longa transcriptome proved instrumental in characterizing the function of the RuBisCO enzyme in this species 23 and enabled identification of a novel Euglenozoa-specific form of the Rheb GTPase 26 , but publication of the whole transcriptome assembly has been pending.…”