Shedding light on the expansion and diversification of the Cdc48 protein family during the rise of the eukaryotic cell.

BACKGROUND: A defining feature of eukaryotic cells is the presence of various distinct membrane-bound compartments with different metabolic roles. Material exchange between most compartments occurs via a sophisticated vesicle trafficking system. This intricate cellular architecture of eukaryotes appears to have emerged suddenly, about 2 billion years ago, from much less complex ancestors. How the eukaryotic cell acquired its internal complexity is poorly understood, partly because no prokaryotic precursors have been found for many key factors involved in compartmentalization. One exception is the Cdc48 protein family, which consists of several distinct classical ATPases associated with various cellular activities (AAA+) proteins with two consecutive AAA domains. RESULTS: Here, we have classified the Cdc48 family through iterative use of hidden Markov models and tree building. We found only one type, Cdc48, in prokaryotes, although a set of eight diverged members that function at distinct subcellular compartments were retrieved from eukaryotes and were probably present in the last eukaryotic common ancestor (LECA). Pronounced changes in sequence and domain structure during the radiation into the LECA set are delineated. Moreover, our analysis brings to light lineage-specific losses and duplications that often reflect important biological changes. Remarkably, we also found evidence for internal duplications within the LECA set that probably occurred during the rise of the eukaryotic cell. CONCLUSIONS: Our analysis corroborates the idea that the diversification of the Cdc48 family is closely intertwined with the development of the compartments of the eukaryotic cell.

Primordial neurosecretory apparatus identified in the choanoflagellate Monosiga brevicollis.

SNARE protein-driven secretion of neurotransmitters from synaptic vesicles is at the center of neuronal communication. In the absence of the cytosolic protein Munc18-1, synaptic secretion comes to a halt. Although it is believed that Munc18-1 orchestrates SNARE complexes, its mode of action is still a matter of debate. In particular, it has been challenging to clarify the role of a tight Munc18/syntaxin 1 complex, because this interaction interferes strongly with syntaxin's ability to form a SNARE complex. In this complex, two regions of syntaxin, the N-peptide and the remainder in closed conformation, bind to Munc18 simultaneously. Until now, this binary complex has been reported for neuronal tissues only, leading to the hypothesis that it might be a specialization of the neuronal secretion apparatus. Here we aimed, by comparing the core secretion machinery of the unicellular choanoflagellate Monosiga brevicollis with that of animals, to reconstruct the ancestral function of the Munc18/syntaxin1 complex. We found that the Munc18/syntaxin 1 complex from M. brevicollis is structurally and functionally highly similar to the vertebrate complex, suggesting that it constitutes a fundamental step in the reaction pathway toward SNARE assembly. We thus propose that the primordial secretion machinery of the common ancestor of choanoflagellates and animals has been co-opted for synaptic roles during the rise of animals.

Quantitative analysis of synaptic vesicle Rabs uncovers distinct yet overlapping roles for Rab3a and Rab27b in Ca2+-triggered exocytosis.

Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca(2+)-triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.

A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex.

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight alpha-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1.SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil "trigger site." This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1.SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.

Differences in the SNARE evolution of fungi and metazoa.

Proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) family are key factors in all vesicle-fusion steps in the endocytic and secretory pathways. SNAREs can assemble into a tight four-helix bundle complex between opposing membranes, a process that is thought to pull the two membranes into close proximity. The complex-forming domains are highly conserved, not only between different species, but also between different vesicular trafficking steps. SNARE protein sequences can be classified into four main types (Qa, Qb, Qc and R), each reflecting their position in the four-helix bundle. Further refinement of these main types resulted in the identification of 20 distinct conserved groups, which probably reflect the original repertoire of a proto-eukaryotic cell. We analysed the evolution of the SNARE repertoires in metazoa and fungi and unveiled remarkable differences in both lineages. In metazoa, the SNARE repertoire appears to have undergone a substantial expansion, particularly in the endosomal pathways. This expansion probably occurred during the transition from a unicellular to a multicellular lifestyle. We also observed another expansion that led to a major increase of the secretory SNAREs in the vertebrate lineage. Interestingly, fungi developed multicellularity independently, but in contrast with plants and metazoa, this change was not accompanied by an expansion of the SNARE set. Our findings suggest that the rise of multicellularity is not generally linked to an expansion of the SNARE set. The structural and functional diversity that exists between fungi and metazoa might offer a simple explanation for the distinct evolutionary history of their SNARE repertoires.

Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi.

BACKGROUND: In eukaryotic cells, directional transport between different compartments of the endomembrane system is mediated by vesicles that bud from a donor organelle and then fuse with an acceptor organelle. A family of integral membrane proteins, termed soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, constitute the key machineries of these different membrane fusion events. Over the past 30 years, the yeast Saccharomyces cerevisiae has served as a powerful model organism for studying the organization of the secretory and endocytic pathways, and a few years ago, its entire set of SNAREs was compiled. RESULTS: Here, we make use of the increasing amount of genomic data to investigate the history of the SNARE family during fungi evolution. Moreover, since different SNARE family members are thought to demarcate different organelles and vesicles, this approach allowed us to compare the organization of the endomembrane systems of yeast and animal cells. Our data corroborate the notion that fungi generally encompass a relatively simple set of SNARE proteins, mostly comprising the SNAREs of the proto-eukaryotic cell. However, all fungi contain a novel soluble SNARE protein, Vam7, which carries an N-terminal PX-domain that acts as a phosphoinositide binding module. In addition, the points in fungal evolution, at which lineage-specific duplications and diversifications occurred, could be determined. For instance, the endosomal syntaxins Pep12 and Vam3 arose from a gene duplication that occurred within the Saccharomycotina clade. CONCLUSION: Although the SNARE repertoire of baker's yeast is highly conserved, our analysis reveals that it is more deviated than the ones of basal fungi. This highlights that the trafficking pathways of baker's yeast are not only different to those in animal cells but also are somewhat different to those of many other fungi.

SNAREing the basis of multicellularity: consequences of protein family expansion during evolution.

Vesicle trafficking between intracellular compartments of eukaryotic cells is mediated by conserved protein machineries. In each trafficking step, fusion of the vesicle with the acceptor membrane is driven by a set of distinctive soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that assemble into tight 4-helix bundle complexes between the fusing membranes. During evolution, about 20 primordial SNARE types were modified independently in different eukaryotic lineages by episodes of duplication and diversification. Here we show that 2 major changes in the SNARE repertoire occurred in the evolution of animals, each reflecting a main overhaul of the endomembrane system. In addition, we found several lineage-specific losses of distinct SNAREs, particularly in nematodes and platyhelminthes. The first major transformation took place during the transition to multicellularity. The primary event that occurred during this transformation was an increase in the numbers of endosomal SNAREs, but the SNARE-related factor lethal giant larvae also emerged. Apparently, enhanced endosomal sorting capabilities were an advantage for early multicellular animals. The second major transformation during the rise of vertebrates resulted in a robust expansion of the secretory set of SNAREs, which may have helped develop a more versatile secretory apparatus.

Drawing explicit phylogenetic networks and their integration into SplitsTree.

BACKGROUND: SplitsTree provides a framework for the calculation of phylogenetic trees and networks. It contains a wide variety of methods for the import/export, calculation and visualization of phylogenetic information. The software is developed in Java and implements a command line tool as well as a graphical user interface. RESULTS: In this article, we present solutions to two important problems in the field of phylogenetic networks. The first problem is the visualization of explicit phylogenetic networks. To solve this, we present a modified version of the equal angle algorithm that naturally integrates reticulations into the layout process and thus leads to an appealing visualization of these networks. The second problem is the availability of explicit phylogenetic network methods for the general user. To advance the usage of explicit phylogenetic networks by biologists further, we present an extension to the SplitsTree framework that integrates these networks. By addressing these two problems, SplitsTree is among the first programs that incorporates implicit and explicit network methods together with standard phylogenetic tree methods in a graphical user interface environment. CONCLUSION: In this article, we presented an extension of SplitsTree 4 that incorporates explicit phylogenetic networks. The extension provides a set of core classes to handle explicit phylogenetic networks and a visualization of these networks.

An elaborate classification of SNARE proteins sheds light on the conservation of the eukaryotic endomembrane system.

Proteins of the SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) family are essential for the fusion of transport vesicles with an acceptor membrane. Despite considerable sequence divergence, their mechanism of action is conserved: heterologous sets assemble into membrane-bridging SNARE complexes, in effect driving membrane fusion. Within the cell, distinct functional SNARE units are involved in different trafficking steps. These functional units are conserved across species and probably reflect the conservation of the particular transport step. Here, we have systematically analyzed SNARE sequences from 145 different species and have established a highly accurate classification for all SNARE proteins. Principally, all SNAREs split into four basic types, reflecting their position in the four-helix bundle complex. Among these four basic types, we established 20 SNARE subclasses that probably represent the original repertoire of a eukaryotic cenancestor. This repertoire has been modulated independently in different lines of organisms. Our data are in line with the notion that the ur-eukaryotic cell was already equipped with the various compartments found in contemporary cells. Possibly, the development of these compartments is closely intertwined with episodes of duplication and divergence of a prototypic SNARE unit.

Computing recombination networks from binary sequences.

MOTIVATION: Phylogenetic networks are becoming an important tool in molecular evolution, as the evolutionary role of reticulate events, such as hybridization, horizontal gene transfer and recombination, is becoming more evident, and as the available data is dramatically increasing in quantity and quality. RESULTS: This paper addresses the problem of computing a most parsimonious recombination network for an alignment of binary sequences that are assumed to have arisen under the 'infinite sites' model of evolution with recombinations. Using the concept of a splits network as the underlying datastructure, this paper shows how a recent method designed for the computation of hybridization networks can be extended to also compute recombination networks. A robust implementation of the approach is provided and is illustrated using a number of real biological datasets. AVAILABILITY: Our implementation of this approach is freely available as part of the SplitsTree4 software, downloadable from www.splitstree.org