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1.  Ancient Complexity, Opisthokont Plasticity, and Discovery of the 11th Subfamily of Arf GAP Proteins 
Traffic (Copenhagen, Denmark)  2013;14(6):636-649.
The organelle paralogy hypothesis is one model for the acquisition of non-endosymbiotic organelles, generated from molecular evolutionary analyses of proteins encoding specificity in the membrane traffic system. GTPase Activating Proteins (GAPs) for the ADP-ribosylation factor (Arfs) GTPases are additional regulators of the kinetics and fidelity of membrane traffic. Here we describe molecular evolutionary analyses of Arf GAP protein family. Of the ten subfamilies previously defined in humans, we find that five were likely present in the Last Eukaryotic Common Ancestor (LECA). Of the three more recently derived subfamilies, one was likely present in the ancestor of opisthokonts (animals and fungi) and apusomonads (flagellates classified as the sister lineage to opisthokonts), while two arose in the holozoan lineage. We also propose to have identified a novel ancient subfamily (ArfGAPC2), present in diverse eukaryotes but which is lost frequently, including in the opisthokonts. Surprisingly few ancient domains accompanying the ArfGAP domain were identified, in marked contrast to the extensively decorated human Arf GAPs. Phylogenetic analyses of the subfamilies reveal patterns of single and multiple gene duplications specific to the Holozoa, to some degree mirroring evolution of Arf GAP targets, the Arfs. Conservation, and lack thereof, of various residues in the ArfGAP structure provide contextualization of previously identified functional amino acids and their application to Arf GAP biology in general. Overall, our results yield insights into current Arf GAP biology, reveal complexity in the ancient eukaryotic ancestor, and integrate the Arf GAP family into a proposed mechanism for the evolution of non-endosymbiotic organelles.
doi:10.1111/tra.12063
PMCID: PMC3660519  PMID: 23433073
membrane traffic; phylogeny; comparative genomics; vesicle transport; GTPase; ADP-ribosylation factors (Arfs); GTPase activating proteins (GAPs); evolutionary cell biology
2.  Characterization of TSET, an ancient and widespread membrane trafficking complex 
eLife  2014;3:e02866.
The heterotetrameric AP and F-COPI complexes help to define the cellular map of modern eukaryotes. To search for related machinery, we developed a structure-based bioinformatics tool, and identified the core subunits of TSET, a 'missing link' between the APs and COPI. Studies in Dictyostelium indicate that TSET is a heterohexamer, with two associated scaffolding proteins. TSET is non-essential in Dictyostelium, but may act in plasma membrane turnover, and is essentially identical to the recently described TPLATE complex, TPC. However, whereas TPC was reported to be plant-specific, we can identify a full or partial complex in every eukaryotic supergroup. An evolutionary path can be deduced from the earliest origins of the heterotetramer/scaffold coat to its multiple manifestations in modern organisms, including the mammalian muniscins, descendants of the TSET medium subunits. Thus, we have uncovered the machinery for an ancient and widespread pathway, which provides new insights into early eukaryotic evolution.
DOI: http://dx.doi.org/10.7554/eLife.02866.001
eLife digest
Eukaryotes make up almost all of the life on Earth that we can see around us, and include organisms as diverse as animals, fungi, plants, slime moulds, and seaweeds. The defining feature of eukaryotes is that, unlike nearly all bacteria, they have membrane-bound compartments—such as the nucleus—within their cells.
Moving molecules, such as proteins, between these compartments is essential for living eukaryotic cells, and these molecules are usually trafficked inside membrane-bound packages called vesicles. Two similar sets of protein complexes—each containing four different subunits—ensure that the molecules are packaged inside the correct vesicles. However, it is not clear how these two protein complexes (called the AP complexes and the COPI complex) are related to each other, and when and where they originated in the history of life.
Now, Hirst, Schlacht et al. have discovered a new—but very ancient–protein complex that they refer to as the ‘missing link’ between the AP and COPI complexes. The four subunits inside this new complex were found by searching for proteins with shapes that were similar to those of the AP and COPI proteins, rather than just searching for proteins with similar sequences of amino acids. This approach identified related protein subunits in groups as diverse as plants and slime moulds, which suggests that this protein complex evolved in the earliest of the eukaryotes. The four subunits identified in a slime mould were confirmed to interact, and also shown to bind to the plasma membrane of living cells.
One of the subunits had already been named TPLATE, so Hirst, Schlacht et al. decided to call the complex TSET; the other three subunits were named TSAUCER, TCUP and TSPOON, and two other proteins that interacted with the complex were both called TTRAY.
While most of the TSET complex itself has been lost from humans and other animals, one of subunit appears to have evolved into a family of proteins that help molecules get into cells. The discovery of TSET reveals another major player in vesicle-trafficking that is not only important for our understanding of how modern eukaryotes work, but also how ancient eukaryotes evolved.
DOI: http://dx.doi.org/10.7554/eLife.02866.002
doi:10.7554/eLife.02866
PMCID: PMC4031984  PMID: 24867644
membrane traffic; clathrin; TPLATE; TPC; muniscin; FCHo; Dictyostelium; human
3.  A comparative analysis of trypanosomatid SNARE proteins☆ 
Parasitology International  2014;63(2):341-348.
The Kinetoplastida are flagellated protozoa evolutionary distant and divergent from yeast and humans. Kinetoplastida include trypanosomatids, and a number of important pathogens. Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. inflict significant morbidity and mortality on humans and livestock as the etiological agents of human African trypanosomiasis, Chagas' disease and leishmaniasis respectively. For all of these organisms, intracellular trafficking is vital for maintenance of the host–pathogen interface, modulation/evasion of host immune system responses and nutrient uptake. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are critical components of the intracellular trafficking machinery in eukaryotes, mediating membrane fusion and contributing to organelle specificity. We asked how the SNARE complement evolved across the trypanosomatids. An in silico search of the predicted proteomes of T. b. brucei and T. cruzi was used to identify candidate SNARE sequences. Phylogenetic analysis, including comparisons with yeast and human SNAREs, allowed assignment of trypanosomatid SNAREs to the Q or R subclass, as well as identification of several SNAREs orthologous with those of opisthokonts. Only limited variation in number and identity of SNAREs was found, with Leishmania major having 27 and T. brucei 26, suggesting a stable SNARE complement post-speciation. Expression analysis of T. brucei SNAREs revealed significant differential expression between mammalian and insect infective forms, especially within R and Qb-SNARE subclasses, suggesting possible roles in adaptation to different environments. For trypanosome SNAREs with clear orthologs in opisthokonts, the subcellular localization of TbVAMP7C is endosomal while both TbSyn5 and TbSyn16B are at the Golgi complex, which suggests conservation of localization and possibly also function. Despite highly distinct life styles, the complement of trypanosomatid SNAREs is quite stable between the three pathogenic lineages, suggesting establishment in the last common ancestor of trypanosomes and Leishmania. Developmental changes to SNARE mRNA levels between blood steam and procyclic life stages suggest that trypanosomes modulate SNARE functions via expression. Finally, the locations of some conserved SNAREs have been retained across the eukaryotic lineage.
Graphical abstract
Highlights
•SNARE proteins are essential components of intracellular transport.•These proteins exhibit considerable conservation across pathogenic trypanosomes.•Some trypanosome SNARE families are expanded or lost.•Developmental changes in trypanosome SNARE expression are apparent.•Orthologous SNAREs demonstrate conserved locations and hence function.
doi:10.1016/j.parint.2013.11.002
PMCID: PMC3979113  PMID: 24269876
Trypanosoma; SNARE; Molecular evolution; Vesicle trafficking
4.  The Mitochondrial Genome and a 60-kb Nuclear DNA Segment from Naegleria fowleri, the Causative Agent of Primary Amoebic Meningoencephalitis 
Naegleria fowleri is a unicellular eukaryote causing primary amoebic meningoencephalitis, a neuropathic disease killing 99% of those infected, usually within 7–14 days. N. fowleri is found globally in regions including the US and Australia. The genome of the related non-pathogenic species Naegleria gruberi has been sequenced, but the genetic basis for N. fowleri pathogenicity is unclear. To generate such insight, we sequenced and assembled the mitochondrial genome and a 60-kb segment of nuclear genome from N. fowleri. The mitochondrial genome is highly similar to its counterpart in N. gruberi in gene complement and organization, while distinct lack of synteny is observed for the nuclear segments. Even in this short (60-kb) segment, we identified examples of potential factors for pathogenesis, including ten novel N. fowleri-specific genes. We also identified a homologue of cathepsin B; proteases proposed to be involved in the pathogenesis of diverse eukaryotic pathogens, including N. fowleri. Finally, we demonstrate a likely case of horizontal gene transfer between N. fowleri and two unrelated amoebae, one of which causes granulomatous amoebic encephalitis. This initial look into the N. fowleri nuclear genome has revealed several examples of potential pathogenesis factors, improving our understanding of a neglected pathogen of increasing global importance.
doi:10.1111/jeu.12022
PMCID: PMC3594069  PMID: 23360210
PAM; meningitis; encephalitis; amoebic infections; infectious disease; genomics; deep sequencing; whole-genome sequencing; amoebic mitochondrial genome
5.  Evolution of Tre-2/Bub2/Cdc16 (TBC) Rab GTPase-activating proteins 
Molecular Biology of the Cell  2013;24(10):1574-1583.
Small GTPases control many functions in cells, and the TBC GTPase-activating protein family modulates the activity of the largest G protein subfamily, Rabs. A reconstruction of the evolutionary history of TBC GAPs provides new insight into the evolution of eukaryotic cells.
Rab GTPases serve as major control elements in the coordination and definition of specific trafficking steps and intracellular compartments. Rab activity is modulated in part by GTPase-activating proteins (GAPs), and many RabGAPs share a Tre-2/Bub2/Cdc16 (TBC)–domain architecture, although the majority of TBC proteins are poorly characterized. We reconstruct the evolutionary history of the TBC family using ScrollSaw, a method for the phylogenetic analysis of pan-eukaryotic data sets, and find a sophisticated, ancient TBC complement of at least 10 members. Significantly, the TBC complement is nearly always smaller than the Rab cohort in any individual genome but also suggests Rab/TBC coevolution. Further, TBC-domain architecture has been well conserved in modern eukaryotes. The reconstruction also shows conservation of ancestral TBC subfamilies, continuing evolution of new TBCs, and frequent secondary losses. These patterns give additional insights into the sculpting of the endomembrane system.
doi:10.1091/mbc.E12-07-0557
PMCID: PMC3655817  PMID: 23485563
6.  Molecular paleontology and complexity in the last eukaryotic common ancestor 
Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself.
doi:10.3109/10409238.2013.821444
PMCID: PMC3791482  PMID: 23895660
Cellular structure; cytoskeleton; endomembrane system; eukaryogenesis; evolution; LECA; metabolomics
7.  Comparative Genomic Analysis of Multi-Subunit Tethering Complexes Demonstrates an Ancient Pan-Eukaryotic Complement and Sculpting in Apicomplexa 
PLoS ONE  2013;8(9):e76278.
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 Guillardiatheta and Bigelowiellanatans. 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.
doi:10.1371/journal.pone.0076278
PMCID: PMC3785458  PMID: 24086721
8.  Evolution and Diversity of the Golgi 
The Golgi is an ancient and fundamental eukaryotic organelle. Evolutionary cell biological studies have begun establishing the repertoire, processes, and level of complexity of membrane-trafficking machinery present in early eukaryotic cells. This article serves as a review of the literature on the topic of Golgi evolution and diversity and reports a novel comparative genomic survey addressing Golgi machinery in the widest taxonomic diversity of eukaryotes sampled to date. Finally, the article is meant to serve as a primer on the rationale and design of evolutionary cell biological studies, hopefully encouraging readers to consider this approach as an addition to their cell biological toolbox. It is clear that the major machinery involved in vesicle trafficking to and from the Golgi was already in place by the time of the divergence of the major eukaryotic lineages, nearly 2 billion years ago. Much of this complexity was likely generated by an evolutionary process involving gene duplication and coevolution of specificity encoding membrane-trafficking proteins. There have also been clear cases of loss of Golgi machinery in some lineages as well as innovation of novel machinery. The Golgi is a wonderfully complex and diverse organelle and its continued exploration promises insight into the evolutionary history of the eukaryotic cell.
The Golgi appeared almost 2 billion years ago, before divergence of the major eukaryotic lineages. Comparative genomics reveals the gene duplication and coevolution of protein specificity that generated its complex trafficking machinery.
doi:10.1101/cshperspect.a007849
PMCID: PMC3140683  PMID: 21646379
9.  A Characterization of the Manduca sexta Serotonin Receptors in the Context of Olfactory Neuromodulation 
PLoS ONE  2013;8(7):e69422.
Neuromodulation, the alteration of individual neuron response properties, has dramatic consequences for neural network function and is a phenomenon observed across all brain regions and taxa. However, the mechanisms underlying neuromodulation are made complex by the diversity of neuromodulatory receptors expressed within a neural network. In this study we begin to examine the receptor basis for serotonergic neuromodulation in the antennal lobe of Manduca sexta. To this end we cloned all four known insect serotonin receptor types from Manduca (the Ms5HTRs). We used phylogenetic analyses to classify the Ms5HTRs and to establish their relationships to other insect serotonin receptors, other insect amine receptors and the vertebrate serotonin receptors. Pharmacological assays demonstrated that each Ms5HTR was selective for serotonin over other endogenous amines and that serotonin had a similar potency at all four Ms5HTRs. The pharmacological assays also identified several agonists and antagonists of the different Ms5HTRs. Finally, we found that the Ms5HT1A receptor was expressed in a subpopulation of GABAergic local interneurons suggesting that the Ms5HTRs are likely expressed heterogeneously within the antennal lobe based on functional neuronal subtype.
doi:10.1371/journal.pone.0069422
PMCID: PMC3726668  PMID: 23922709
11.  Correction: The Fifth Adaptor Protein Complex 
PLoS Biology  2012;10(3):10.1371/annotation/89dff893-c156-44bb-a731-bfcc91843583.
doi:10.1371/annotation/89dff893-c156-44bb-a731-bfcc91843583
PMCID: PMC3312919
12.  Correction: A Novel Rho-Like Protein TbRHP Is Involved in Spindle Formation and Mitosis in Trypanosomes 
PLoS ONE  2011;6(11):10.1371/annotation/b4b38a99-0b4c-483a-ad21-4dd721974b97.
doi:10.1371/annotation/b4b38a99-0b4c-483a-ad21-4dd721974b97
PMCID: PMC3228669
13.  A Novel Rho-Like Protein TbRHP Is Involved in Spindle Formation and Mitosis in Trypanosomes 
PLoS ONE  2011;6(11):e26890.
Background
In animals and fungi Rho subfamily small GTPases are involved in signal transduction, cytoskeletal function and cellular proliferation. These organisms typically possess multiple Rho paralogues and numerous downstream effectors, consistent with the highly complex contributions of Rho proteins to cellular physiology. By contrast, trypanosomatids have a much simpler Rho-signaling system, and the Trypanosoma brucei genome contains only a single divergent Rho-related gene, TbRHP (Tb927.10.6240). Further, only a single RhoGAP-like protein (Tb09.160.4180) is annotated, contrasting with the >70 Rho GAP proteins from Homo sapiens. We wished to establish the function(s) of TbRHP and if Tb09.160.4180 is a potential GAP for this protein.
Methods/Findings
TbRHP represents an evolutionarily restricted member of the Rho GTPase clade and is likely trypanosomatid restricted. TbRHP is expressed in both mammalian and insect dwelling stages of T. brucei and presents with a diffuse cytoplasmic location and is excluded from the nucleus. RNAi ablation of TbRHP results in major cell cycle defects and accumulation of multi-nucleated cells, coinciding with a loss of detectable mitotic spindles. Using yeast two hybrid analysis we find that TbRHP interacts with both Tb11.01.3180 (TbRACK), a homolog of Rho-kinase, and the sole trypanosome RhoGAP protein Tb09.160.4180, which is related to human OCRL.
Conclusions
Despite minimization of the Rho pathway, TbRHP retains an important role in spindle formation, and hence mitosis, in trypanosomes. TbRHP is a partner for TbRACK and an OCRL-related trypanosome Rho-GAP.
doi:10.1371/journal.pone.0026890
PMCID: PMC3214021  PMID: 22096505
14.  The Fifth Adaptor Protein Complex 
PLoS Biology  2011;9(10):e1001170.
Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.
Author Summary
Adaptor protein (AP) complexes facilitate the trafficking of cargo from one membrane compartment of the cell to another by recruiting other proteins to particular types of vesicles. For over 10 years, it has been assumed that there are four, and only four, distinct AP complexes in eukaryotic cells. We report the existence of a fifth AP complex, AP-5. Immunolocalisation and RNAi knockdown experiments both indicate that AP-5 is involved in trafficking proteins from endosomes towards other membranous compartments. There are genetic links between AP-5 and hereditary spastic paraplegia, a group of human genetic disorders characterised by progressive spasticity in the lower limbs. Phylogenetic analyses indicate that AP-5 was already present in the last eukaryotic common ancestor over a billion years ago.
doi:10.1371/journal.pbio.1001170
PMCID: PMC3191125  PMID: 22022230
15.  Evolution of the Karyopherin-β Family of Nucleocytoplasmic Transport Factors; Ancient Origins and Continued Specialization 
PLoS ONE  2011;6(4):e19308.
Background
Macromolecular transport across the nuclear envelope (NE) is achieved through nuclear pore complexes (NPCs) and requires karyopherin-βs (KAP-βs), a family of soluble receptors, for recognition of embedded transport signals within cargo. We recently demonstrated, through proteomic analysis of trypanosomes, that NPC architecture is likely highly conserved across the Eukaryota, which in turn suggests conservation of the transport mechanisms. To determine if KAP-β diversity was similarly established early in eukaryotic evolution or if it was subsequently layered onto a conserved NPC, we chose to identify KAP-β sequences in a diverse range of eukaryotes and to investigate their evolutionary history.
Results
Thirty six predicted proteomes were scanned for candidate KAP-β family members. These resulting sequences were resolved into fifteen KAP-β subfamilies which, due to broad supergroup representation, were most likely represented in the last eukaryotic common ancestor (LECA). Candidate members of each KAP-β subfamily were found in all eukaryotic supergroups, except XPO6, which is absent from Archaeplastida. Phylogenetic reconstruction revealed the likely evolutionary relationships between these different subfamilies. Many species contain more than one representative of each KAP-β subfamily; many duplications are apparently taxon-specific but others result from duplications occurring earlier in eukaryotic history.
Conclusions
At least fifteen KAP-β subfamilies were established early in eukaryote evolution and likely before the LECA. In addition we identified expansions at multiple stages within eukaryote evolution, including a multicellular plant-specific KAP-β, together with frequent secondary losses. Taken with evidence for early establishment of NPC architecture, these data demonstrate that multiple pathways for nucleocytoplasmic transport were established prior to the radiation of modern eukaryotes but that selective pressure continues to sculpt the KAP-β family.
doi:10.1371/journal.pone.0019308
PMCID: PMC3083441  PMID: 21556326
16.  Arf3 Is Activated Uniquely at the trans-Golgi Network by Brefeldin A-inhibited Guanine Nucleotide Exchange Factors 
Molecular Biology of the Cell  2010;21(11):1836-1849.
Arf3 associates with the TGN in a manner that is both temperature-sensitive and uniquely dependent on BIGs. TGN localization and release at 20°C are readily separated and depend on pairs of residues absolutely conserved and unique to Arf3 present at opposite ends of the protein. These results suggest that Arf3 plays a unique function at the TGN.
It is widely assumed that class I and II Arfs function interchangeably throughout the Golgi complex. However, we report here that in vivo, Arf3 displays several unexpected properties. Unlike other Golgi-localized Arfs, Arf3 associates selectively with membranes of the trans-Golgi network (TGN) in a manner that is both temperature-sensitive and uniquely dependent on guanine nucleotide exchange factors of the BIGs family. For example, BIGs knockdown redistributed Arf3 but not Arf1 from Golgi membranes. Furthermore, shifting temperature to 20°C, a temperature known to block cargo in the TGN, selectively redistributed Arf3 from Golgi membranes. Arf3 redistribution occurred slowly, suggesting it resulted from a change in membrane composition. Arf3 knockdown and overexpression experiments suggest that redistribution is not responsible for the 20°C block. To investigate in more detail the mechanism for Arf3 recruitment and temperature-dependent release, we characterized several mutant forms of Arf3. This analysis demonstrated that those properties are readily separated and depend on pairs of residues present at opposite ends of the protein. Furthermore, phylogenetic analysis established that all four critical residues were absolutely conserved and unique to Arf3. These results suggest that Arf3 plays a unique function at the TGN that likely involves recruitment by a specific receptor.
doi:10.1091/mbc.E10-01-0016
PMCID: PMC2877642  PMID: 20357002
17.  Rab protein evolution and the history of the eukaryotic endomembrane system 
Cellular and Molecular Life Sciences  2010;67(20):3449-3465.
Spectacular increases in the quantity of sequence data genome have facilitated major advances in eukaryotic comparative genomics. By exploiting homology with classical model organisms, this makes possible predictions of pathways and cellular functions currently impossible to address in intractable organisms. Echoing realization that core metabolic processes were established very early following evolution of life on earth, it is now emerging that many eukaryotic cellular features, including the endomembrane system, are ancient and organized around near-universal principles. Rab proteins are key mediators of vesicle transport and specificity, and via the presence of multiple paralogues, alterations in interaction specificity and modification of pathways, contribute greatly to the evolution of complexity of membrane transport. Understanding system-level contributions of Rab proteins to evolutionary history provides insight into the multiple processes sculpting cellular transport pathways and the exciting challenges that we face in delving further into the origins of membrane trafficking specificity.
doi:10.1007/s00018-010-0436-1
PMCID: PMC2943070  PMID: 20582450
Trafficking; Evolution; Eukaryogenesis; Rab protein; Systems biology
18.  Pex3 peroxisome biogenesis proteins function in peroxisome inheritance as class V myosin receptors 
The Journal of Cell Biology  2009;187(2):233-246.
Pex3 links peroxisome formation and inheritance. By binding to class V myosin, biogenesis protein Pex3 also directs the organelles into daughter cells.
In Saccharomyces cerevisiae, peroxisomal inheritance from mother cell to bud is conducted by the class V myosin motor, Myo2p. However, homologues of S. cerevisiae Myo2p peroxisomal receptor, Inp2p, are not readily identifiable outside the Saccharomycetaceae family. Here, we demonstrate an unexpected role for Pex3 proteins in peroxisome inheritance. Both Pex3p and Pex3Bp are peroxisomal integral membrane proteins that function as peroxisomal receptors for class V myosin through direct interaction with the myosin globular tail. In cells lacking Pex3Bp, peroxisomes are preferentially retained by the mother cell, whereas most peroxisomes gather and are transferred en masse to the bud in cells overexpressing Pex3Bp or Pex3p. Our results reveal an unprecedented role for members of the Pex3 protein family in peroxisome motility and inheritance in addition to their well-established role in peroxisome biogenesis at the endoplasmic reticulum. Our results point to a temporal link between peroxisome formation and inheritance and delineate a general mechanism of peroxisome inheritance in eukaryotic cells.
doi:10.1083/jcb.200902117
PMCID: PMC2768826  PMID: 19822674
19.  Comparative analysis of plant genomes allows the definition of the "Phytolongins": a novel non-SNARE longin domain protein family 
BMC Genomics  2009;10:510.
Background
Subcellular trafficking is a hallmark of eukaryotic cells. Because of their pivotal role in the process, a great deal of attention has been paid to the SNARE proteins. Most R-SNAREs, or "longins", however, also possess a highly conserved, N-terminal fold. This "longin domain" is known to play multiple roles in regulating SNARE activity and targeting via interaction with other trafficking proteins. However, the diversity and complement of longins in eukaryotes is poorly understood.
Results
Our comparative genome survey identified a novel family of longin-related proteins, dubbed the "Phytolongins" because they are specific to land plants. Phytolongins share with longins the N-terminal longin domain and the C-terminal transmembrane domain; however, in the central region, the SNARE motif is replaced by a novel region. Phylogenetic analysis pinpoints the Phytolongins as a derivative of the plant specific VAMP72 longin sub-family and allows elucidation of Phytolongin evolution.
Conclusion
"Longins" have been defined as R-SNAREs composed of both a longin domain and a SNARE motif. However, expressed gene isoforms and splice variants of longins are examples of non-SNARE motif containing longins. The discovery of Phytolongins, a family of non-SNARE longin domain proteins, together with recent evidence on the conservation of the longin-like fold in proteins involved in both vesicle fusion (e.g. the Trs20 tether) and vesicle formation (e.g. σ and μ adaptin) highlight the importance of the longin-like domain in protein trafficking and suggest that it was one of the primordial building blocks of the eukaryotic membrane-trafficking machinery.
doi:10.1186/1471-2164-10-510
PMCID: PMC2779197  PMID: 19889231
20.  The cloning of one putative octopamine receptor and two putative serotonin receptors from the tobacco hawkmoth, Manduca sexta 
Serotonin and octopamine (OA) are biogenic amines that are active throughout the nervous systems of insects, affecting sensory processing, information coding and behavior. As an initial step towards understanding the modulatory roles of these amines in olfactory processing we cloned two putative serotonin receptors (Ms5HT1A and Ms5HT1B) and one putative OA (MsOAR) receptor from the moth Manduca sexta. Ms5HT1A and Ms5HT1B were both similar to 5HT1-type receptors but differed from each other in their N-terminus and 3rd cytoplasmic loop. Ms5HT1A was nearly identical to a serotonin receptor from Heliothis virescens and Ms5HT1B was almost identical to a serotonin receptor from Bombyx mori. The sequences for homologs of Ms5HT1A from B. mori and Ms5HT1B from H. virescens were also obtained, suggesting that the Lepidoptera likely have at least two serotonin receptors. The MsOAR shares significant sequence homology with pharmacologically characterized OA receptors, but less similarity to putative OA/tyramine receptors from the moths B. mori and H. virescens. Using the MsOAR sequence, fragments encoding putative OA receptors were obtained from B. mori and H. virescens, suggesting that MsOAR is the first OA receptor cloned from a lepidopteran.
doi:10.1016/j.ibmb.2006.07.002
PMCID: PMC1794002  PMID: 16935223
Serotonin; Octopamine; Manduca; Receptors; Modulation
21.  Draft Genome Sequence of the Sexually Transmitted Pathogen Trichomonas vaginalis 
Science (New York, N.Y.)  2007;315(5809):207-212.
We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the ~160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.
doi:10.1126/science.1132894
PMCID: PMC2080659  PMID: 17218520
22.  Reconstructing the Mosaic Glycolytic Pathway of the Anaerobic Eukaryote Monocercomonoides▿ †  
Eukaryotic Cell  2006;5(12):2138-2146.
All eukaryotes carry out glycolysis, interestingly, not all using the same enzymes. Anaerobic eukaryotes face the challenge of fewer molecules of ATP extracted per molecule of glucose due to their lack of a complete tricarboxylic acid cycle. This may have pressured anaerobic eukaryotes to acquire the more ATP-efficient alternative glycolytic enzymes, such as pyrophosphate-fructose 6-phosphate phosphotransferase and pyruvate orthophosphate dikinase, through lateral gene transfers from bacteria and other eukaryotes. Most studies of these enzymes in eukaryotes involve pathogenic anaerobes; Monocercomonoides, an oxymonad belonging to the eukaryotic supergroup Excavata, is a nonpathogenic anaerobe representing an evolutionarily and ecologically distinct sampling of an anaerobic glycolytic pathway. We sequenced cDNA encoding glycolytic enzymes from a previously established cDNA library of Monocercomonoides and analyzed the relationships of these enzymes to those from other organisms spanning the major groups of Eukaryota, Bacteria, and Archaea. We established that, firstly, Monocercomonoides possesses alternative versions of glycolytic enzymes: fructose-6-phosphate phosphotransferase, both pyruvate kinase and pyruvate orthophosphate dikinase, cofactor-independent phosphoglycerate mutase, and fructose-bisphosphate aldolase (class II, type B). Secondly, we found evidence for the monophyly of oxymonads, kinetoplastids, diplomonads, and parabasalids, the major representatives of the Excavata. We also found several prokaryote-to-eukaryote as well as eukaryote-to-eukaryote lateral gene transfers involving glycolytic enzymes from anaerobic eukaryotes, further suggesting that lateral gene transfer was an important factor in the evolution of this pathway for denizens of this environment.
doi:10.1128/EC.00258-06
PMCID: PMC1694820  PMID: 17071828
23.  Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins 
Background
In membrane trafficking, the mechanisms ensuring vesicle fusion specificity remain to be fully elucidated. Early models proposed that specificity was encoded entirely by SNARE proteins; more recent models include contributions from Rab proteins, Syntaxin-binding (SM) proteins and tethering factors. Most information on membrane trafficking derives from an evolutionarily narrow sampling of model organisms. However, considering factors from a wider diversity of eukaryotes can provide both functional information on core systems and insight into the evolutionary history of the trafficking machinery. For example, the major Qa/syntaxin SNARE families are present in most eukaryotic genomes and likely each evolved via gene duplication from a single ancestral syntaxin before the existing eukaryotic groups diversified. This pattern is also likely for Rabs and various other components of the membrane trafficking machinery.
Results
We performed comparative genomic and phylogenetic analyses, when relevant, on the SM proteins and components of the tethering complexes, both thought to contribute to vesicle fusion specificity. Despite evidence suggestive of secondary losses amongst many lineages, the tethering complexes are well represented across the eukaryotes, suggesting an origin predating the radiation of eukaryotic lineages. Further, whilst we detect distant sequence relations between GARP, COG, exocyst and DSL1 components, these similarities most likely reflect convergent evolution of similar secondary structural elements. No similarity is found between the TRAPP and HOPS complexes and the other tethering factors. Overall, our data favour independent origins for the various tethering complexes. The taxa examined possess at least one homologue of each of the four SM protein families; since the four monophyletic families each encompass a wide diversity of eukaryotes, the SM protein families very likely evolved before the last common eukaryotic ancestor (LCEA).
Conclusion
These data further support a highly complex LCEA and indicate that the basic architecture of the trafficking system is remarkably conserved and ancient, with the SM proteins and tethering factors having originated very early in eukaryotic evolution. However, the independent origin of the tethering complexes suggests a novel pattern for increasing complexity in the membrane trafficking system, in addition to the pattern of paralogous machinery elaboration seen thus far.
doi:10.1186/1471-2148-7-29
PMCID: PMC1810245  PMID: 17319956
24.  Evolutionary Origins of the Eukaryotic Shikimate Pathway: Gene Fusions, Horizontal Gene Transfer, and Endosymbiotic Replacements†  
Eukaryotic Cell  2006;5(9):1517-1531.
Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been an important process in eukaryote genome evolution.
doi:10.1128/EC.00106-06
PMCID: PMC1563581  PMID: 16963634
25.  Evidence for Golgi bodies in proposed 'Golgi-lacking' lineages. 
Golgi bodies are nearly ubiquitous in eukaryotic cells. The apparent lack of such structures in certain eukaryotic lineages might be taken to mean that these protists evolved prior to the acquisition of the Golgi, and it raises questions of how these organisms function in the absence of this crucial organelle. Here, we report gene sequences from five proposed 'Golgi-lacking' organisms (Giardia intestinalis, Spironucleus barkhanus, Entamoeba histolytica, Naegleria gruberi and Mastigamoeba balamuthi). BLAST and phylogenetic analyses show these genes to be homologous to those encoding components of the retromer, coatomer and adaptin complexes, all of which have Golgi-related functions in mammals and yeast. This is, to our knowledge, the first molecular evidence for Golgi bodies in two major eukaryotic lineages (the pelobionts and heteroloboseids). This substantiates the suggestion that there are no extant primitively 'Golgi-lacking' lineages, and that this apparatus was present in the last common eukaryotic ancestor, but has been altered beyond recognition several times.
doi:10.1098/rsbl.2003.0058
PMCID: PMC1809936  PMID: 14667372

Results 1-25 (25)