Multivesicular bodies in the enigmatic amoeboflagellate Breviata anathema and the evolution of ESCRT 0

Emily K. Herman 2 Giselle Walker 1 Mark van der Giezen 0 Joel B. Dacks () 2 0 Centre for Eukaryotic Evolutionary Microbiology , Biosciences , College of Life and Environmental Sciences, University of Exeter , Exeter EX4 4QD , UK 1 Department of Earth Sciences, University of Cambridge , Downing Street, Cambridge CB2 3EQ , UK 2 Department of Cell Biology, School of Molecular and Systems Medicine, Faculty of Medicine and Dentistry, University of Alberta , Edmonton, AB T6G 2H7 , Canada - Summary Endosomal sorting complexes required for transport (ESCRTs) are heteromeric protein complexes required for multivesicular body (MVB) morphogenesis. ESCRTs I, II, III and III-associated are ubiquitous in eukaryotes and presumably ancient in origin. ESCRT 0 recruits cargo to the MVB and appears to be opisthokont-specific, bringing into question aspects of the current model of ESCRT ce mechanism. One caveat to the restricted distribution of ESCRT 0 was the previous limited availability of amoebozoan genomes, the n supergroup closest to opisthokonts. Here, we significantly expand the sampling of ESCRTs in Amoebozoa. Our electron micrographic e ic and bioinformatics evidence confirm the presence of MVBs in the amoeboflagellate Breviata anathema. Searches of genomic databases S of amoebozoans confirm the ubiquitous nature of ESCRTs IIII-associated and the restriction of ESCRT 0 to opisthokonts. Recently, lle an alternate ESCRT 0 complex, centering on Tom1 proteins, has been proposed. We determine the distribution of Tom1 family proteins C across eukaryotes and show that the Tom1, Tom1L1 and Tom1L2 proteins are a vertebrate-specific expansion of the single Tom1 family fo ancestor, which has indeed been identified in at least one member of each of the major eukaryotic supergroups. This implies a more l widely conserved and ancient role for the Tom1 family in endocytosis than previously suspected. a n r u Key words: Endocytosis, Eukaryote, Membrane trafficking o J Introduction Multivesicular bodies (MVBs) are crucial eukaryotic organelles involved in ubiquitin-mediated endocytic processes, underlying cellular acquisition of nutrients, and downregulation of receptors (Williams and Urbe, 2007). They are generally sized at 400500 nm (Gruenberg and Stenmark, 2004) and contain intraluminal vesicles (ILVs) that are uniformly round and 50 nm in diameter or smaller (Williams and Urbe, 2007). ILVs are created by the invagination and inward budding of the membrane, a process modulated by a set of protein components collectively known as the endosomal sorting complexes required for transport, or ESCRTs. There are five soluble ESCRT complexes that work at the cytosolic face of the MVB and are responsible for recruiting the proper cargo to ILVs, as well as the budding and scission events (Hurley, 2008). An emerging model of ESCRT function (Fig. 1) in mammalian and yeast systems identifies the ESCRT 0 complex as recognizing and binding ubiquitylated cargo from sites such as the plasma membrane and recruiting it to the MVB. The mechanism of this process is unclear because a recent study has shown that depletion of the human Vps27 (an ESCRT 0 component) by siRNA does not have a significant effect on epidermal growth factor receptor (EGFR) endocytosis (Raiborg et al., 2008). Interaction between the ESCRT I component Vps23 with the P[S/T]xP domain in Vps27 recruits ESCRT I to MVBs where it is responsible for cargo sorting and recruitment of ESCRTs II and III. These subcomplexes interact with each other, as well as with the membrane, to mediate inward budding. The ESCRT III-associated machinery has been shown to induce scission of the vesicle (Wollert and Hurley, 2010) and one particular component, the AAA-type ATPase Vps4, is responsible for disassembly of the other ESCRTs (Saksena et al., 2009). The ESCRT machinery is not only functionally essential but ancient as well. Comparative genomic studies have shown that the vast majority of protein components composing ESCRT complexes IIII-associated are present in the diversity of eukaryotic taxa (Field et al., 2007; Leung et al., 2008; Slater and Bishop, 2006), well beyond the model systems of yeast and Metazoa. This implies that the last eukaryotic common ancestor (LECA) possessed an ESCRT machinery of near-modern complexity. Comparative experimental characterization in organisms from various eukaryotic supergroups (Adl et al., 2005) suggests similarity and conservation of ESCRT function as well, with organelles resembling MVBs identified in diverse eukaryotes (Haas et al., 2007; Hurley, 2008; Leung et al., 2008; Yang et al., 2004). Delving further back in evolutionary time, it is apparent that gene duplications gave rise to two sets of components in the ESCRT III and III-associated machinery, the Vps20/Vps32/Vps60 and the Vps2/Vps24/Vps46 families (Leung et al., 2008). This implies a model of an ancestral dimeric ESCRT III complex composed of a progenitor protein from each family (Leung et al., 2008). Such a model is bolstered Fig. 1. Model of ESCRT complex assembly. The diagram is based loosely upon one previously published (Raiborg and Stenmark, 2009) incorporating information from other sources (Hurley, 2008; Im et al., 2009; Kostelansky et al., 2007; Prag et al., 2007; Shestakova et al., 2010; Xiao et al., 2008). The complexes are marked as follows: ESCRT 0 components in red, ESCRT I in green, ESCRT II in orange, ESCRT III in purple, and ESCRT III-associated in blue. Relevant domains are identified. The GAT domain of ESCRT 0 is a heterodimer due to domainswapping. The multicoloured pyramids indicate binding sites of ubiquitylated cargo, and the lipids (yellow circles with tails) indicate phosphatidylinositol 3-phosphate binding sites. The Tom1 complex marked in grey is based on the ancestral complex proposed previously (Blanc et al., 2009). by the recent discovery of ESCRT III homologs in Archaea that are involved in cell division (Samson et al., 2008), providing a path of origin for the ESCRT machinery in eukaryotes (Field and Dacks, 2009). By contrast, ESCRT 0 components appear to be opisthokontspecific (Field et al., 2007; Leung et al., 2008), raising questions ce of the origin of this machinery and the generality of the current en model of ESCRT mechanism. However, at the time of the most ic recent and exhaustive comparative genomic analysis of the ESCRT S machinery to date (Leung et al., 2008), only a limited number of lle genomes were available from the nearest supergroup to the C Opisthokonta, i.e. the Amoebozoa. The possibility therefore exists fo that undersampling might explain the ESCRT 0 distribution. We l have therefore undertaken an investigation of ESCRT machinery a rnu iBnretvhiaetaAamnoaethbeomzoaa., with emphasis on the enigmatic amoeba o J Originally mis-identified as the pelobiont Mastigamoeba invertens, B. anathema is an amoeboid flagellate (Fig. 2A), 510 m in size (Walker et al., 2006). It lacks canonical mitochondria, posse (...truncated)