Comparative Genomic Analysis of Multi-Subunit Tethering Complexes Demonstrates an Ancient Pan-Eukaryotic Complement and Sculpting in Apicomplexa

Abstract

Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs), factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardia theta and Bigelowiella natans . The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during, and possibly pre-dating, the move from free-living marine algae to deadly human parasites.

Mammalian CORVET Is Required for Fusion and Conversion of Distinct Early Endosome Subpopulations

“…Our bioinformatics analysis ( Figure 1A) assigned Tgfbrap1 to either Vps3p/CORVET or Vps39p/HOPS. This experiment and a previous bioinformatics study (35) indicated that Tgfbrap1 could be the functional ortholog of Vps3p. To further support this finding, we sought to validate the interaction with Rab5-GTPγS using a different source of cytosol.…”

“…For co-localization experiments, samples were analyzed using a Laser Scanning Confocal Microscope (Olympus Fluoview 1000) equipped with an Olympus UPlanSApo 60 × 1. 35 Oil immersion objective at a resolution of 100 nm/pixel. siRNA experiments were acquired as described in 45.…”

“…D) HeLa cells labelled by a 3-min pulse of 10 μg/mL Alexa647-Transferrin, were chased and fixed at 4, 5, 7.5, 10, 15, 20, 30, and 45 min (counting from the beginning of pulse, as the levels at 30 and 45 min are close to background, we removed them to improve the visualization of the first time-points). E) HeLa cells labelled by a 15 min pulse of 10 μg/mL LDL were fixed at5,10,15,20,25,35,45, 60, and 75 min (counting from the beginning of pulse, chase starts after 15 min). The vesicular intensity of the cargoes is quantified in the whole cell (total), or on AA, AE, and EE endosomes in a Mock condition (black line), or upon knockdown of Tgfbrap1 (red line) or Vps39 (blue line).…”

Missing Pieces of an Ancient Puzzle: Evolution of the Eukaryotic Membrane-Trafficking System

“…It is seen when investigating many key trafficking complexes ( Fig. 2A) such as the late endosome ESCRTs (Leung et al 2008), and the MTCs (Koumandou et al 2007;Klinger et al 2013), as well as much of the membrane deformation machinery, for example, COP and clathrin coat complexes (Dacks and Field 2004;Neumann et al 2010).…”

Ca2+-regulated secretory granule exocytosis in pancreatic and parotid acinar cells

“…A number of potential tethering proteins are present on acinar SGs including Rab3D [21], Rab27B and the synaptotagmin-like proteins (see below). The molecular details of tethering complexes for constitutive membrane fusion in endosomal and secretory pathways are still emerging with some such as the exocyst, HOPS, and CORVET involving large multimeric protein complexes [6, 22, 23]. A recent study in parotid acini identified subunits of the multimeric exocyst tethering complex along the apical membrane and provided the first evidence for a role of a multimeric tethering complex in SG exocytosis by demonstrating the inhibition of isoproterenol-induced amylase secretion following introduction of subunit-specific antibodies into permeabilized acinar cells [24].…”