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

<p>Abstract</p> <p>Background</p> <p>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 <it>Saccharomyces cerevisiae </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. RESULTS: Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. CONCLUSIONS: Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types

Phylogenetische Studien der vesikulären Fusionsmaschinerie

Die eukaryotische Zelle ist in mehrere Kompartimente unterteilt, welche durch Membranen vom Rest der Zelle abgetrennt sind. Stoffaustausch zwischen Kompartimenten geschieht über vesikulären Transport. Vesikel werden am Donorkompartment abgeschnürt, wandern danach entlang des Zytoskeletts, um schliesslich mit der Membran des Zielkompartiments zu fusionieren. Verschiedene Proteinfamilien (z.B. SNARE, SNAP, Rab, SM, C2 Domänen Proteine), die im intrazellulären Transport eine entscheidende Rolle spielen, sind hoch konserviert. Dies gilt nicht nur für unterschiedliche Organismen, sondern auch für die unterschiedlichen Transportschritte innerhalb einer Zelle. Die genau Funktionsweise dieser Proteinfamilien ist häufig unklar und es existieren wenig Hinweise auf deren evolutionärer Entwicklung. Eine Untersuchung der Entstehung dieser Proteinfamilien könnte interessante Einblicke in die molekularen Ereignisse liefern. Zur Durchführung einer solchen Untersuchung bedarf es einiger technischer Voraussetzungen. Die vorliegende Arbeit beschreibt die Entwicklung des flexiblen und leistungsfähigen Tracey Verwaltungssystems (Datenbank, Java Datenbankpaket und Webseite). Dieses innovative System erlaubt die Klassifizierung und Analyse selbst von hoch komplexen und mannigfaltigen Proteinfamilien. Darüber hinaus wurde das System eingesetzt, um die SNARE Proteine in Pilzen, die SNAP Familie und die C2 Domänen in Pilzen zu untersuchen

Back to the future?: Can Europe meet its labour needs through temporary migration?

Around 1974, most Western European countries abandoned policies of migrant labour recruitment, and moved towards increasingly restrictive entry rules. Today, employers, politicians and European Commission officials are considering a return to policies of systematic admission of migrant workers. Temporary or seasonal migrant worker programs have already been introduced in a number of countries. This paper inquires whether Europe is likely to return to pre-1974 migrant labour approaches. It examines the demographic, economic and social changes that have led to this new interest in labour immigration, and looks at recent proposals on temporary migration, including those of the Global Commission on International Migration. It discusses experiences of temporary migrant worker programs in Germany and the UK, and goes on to look at the European Commission's 2005 Policy Plan for Legal Migration. The paper shows that the current approaches differ significantly from the guestworker programs of the past and that there is thus no question of a general return to pre-1974 type policies. However, some current approaches do share important common features with past guestworker programs. They may lead to negative social outcomes in both receiving and sending countries

Additional file 1: Figure S1. of Shedding light on the expansion and diversification of the Cdc48 protein family during the rise of the eukaryotic cell

Statistical validation of the AAA domain classification. (A) We used a resampling approach to evaluate the quality of our Hidden Markov Models (HMMs). New models were trained with a random subset of 90 % of the original sequences used to generate each model. We used the other 10 % as the search database with a fixed size of 100,000 sequences. This process was repeated 1000 times and we considered the profile with the best expectation value to be correct. The positive predictive rate (PPR, black, left) and the sensitivity (white, right) are displayed. All models achieved at least 97 % PPR and sensitivity. (B-D) The Cdc48 family is part of a superfamily of classical AAA proteins that also includes proteasome subunits, metalloproteases, meiotic ATPases, and BCS1 [14]. As all our models were trained using Cdc48 AAA domain sequences, non-Cdc48 AAA domain sequences should be a much weaker fit to these models. To evaluate the specificity of our HMMs, we tested the extent to which our models also recognized non-Cdc48 AAA domains. For this, we selected approximately 1800 sequences from the larger family of classical AAA proteins and scanned these sequences with our models. The results are shown as box plots, including the 5 % and 95 % percentiles as whiskers. The plots show the scores (negative logarithm of the expectation value) of our models for the predictions of (B) Cdc48 sequences and (D) non-Cdc48 sequences. We used the different E-value distributions to define the cut-offs for the confidence of our Cdc48 AAA domain predictions. The 5 % percentile of the expectation value distribution in (B) was used as a ‘strict’ cut-off, whereas the 95 % percentile of the expectation value distribution from (D) served as a ‘soft’ cut-off. An overview of the ‘strict’ versus ’soft’ cut-offs for all Cdc48 domain models are displayed in (C). SPAF.d1 is the only model that reveals a lower ‘strict’ than ‘soft’ cut-off. The ‘strict’ cut-off for this model seems especially low, whereas the ‘soft’ cut-off is in a similar range to most other models. Plotting the scores of predictions on Cdc48 sequences for each AAA domain model reveals that most graphs have a logarithmic characteristic, whereas SPAF.d1 follows a linear trend (data not shown). This indicates a higher degree of diversity within this domain. To uphold the quality of our predictions we decided to use the higher ‘soft’ cut-off as the ‘strict’ cut-off for SPAF.d1 as well. (AI 4 mb

Additional file 6: Figure S4. of Shedding light on the expansion and diversification of the Cdc48 protein family during the rise of the eukaryotic cell

A detailed view of the tail helix region of Cdc48. (A) WebLogo representation [119] of the tail region of different Cdc48 family members. Note that the C-terminal HbYX motif of Cdc48 is not maintained in other family members, with the possible exception of Pex1. (B) The structure of the tail region of Cdc48 (PDB: 3CF1 [43]). In the tail helix (in yellow) of Cdc48, the residue Y755 contacts the sensor 1 residue N624. Unfortunately, the structure of the D2 domain of human nuclear VCP-like (NVL, (PDB ID: 2X8A) does not include the tail helix and therefore it cannot be seen whether its tyrosine also interacts with the Sensor 1 asparagine. (AI 5 mb

Additional file 12: Figure S8. of Shedding light on the expansion and diversification of the Cdc48 protein family during the rise of the eukaryotic cell

WebLogo representation of the two D-domains of the different Cdc48 family members. Sequence logos were generated from alignments of the D-domains of different Cdc48 family members from more than 500 eukaryotes using WebLogo software [119] (see Fig. 3). The overall height of a stack indicates the sequence conservation at a certain position, whereas the height of symbols within the stack indicates the relative frequency of each amino acid at that position. (AI 11 mb

Additional file 3: Figure S2. of Shedding light on the expansion and diversification of the Cdc48 protein family during the rise of the eukaryotic cell

Structural elements of the tandem D-domains of Cdc48. The two D-domains of Cdc48 are formed by two subdomains. (A) The N-terminal αβ subdomain contains various motifs like the Walker A motif (P-loop), the Walker B motif, and the polar Sensor 1 residue, which are important for ATP binding and hydrolysis. The conserved arginine residues at the end of α4, referred to as the Arg finger, are in proximity to the γ-phosphate of the bound ATP in the neighboring subunit. Note that the subunits are active only as hexameric assemblies, a key feature of this protein superfamily. The Cdc48 family belongs to the clade of classical AAA proteins that have a small helical insertion before helix α2 within the Rossman fold [14, 15, 18–20, 22]. The C-terminal subdomain is α-helical. A stretch after Helix α7 that was not resolved in the structure is shown as a dashed line. The base of Helix α7 comprises the Sensor 2 region. Both D-domains of Cdc48 possess a conserved GAD motif in this region. The Sensor 2 aspartate of the D1-domain contacts a conserved stretch at the base of the D1-D2 linker and might be important for communication between the two D-domains (Additional file 5: Figure S3). The Sensor 2 aspartate of the D2-domain of Cdc48 interacts with a stretch in front of the C-terminal helix and thus might help to position this helix [43, 45]. The tail helix is followed by a C-terminal extension with a penultimate HbYX motif (Additional file 6: Figure S4). Usually, this motif is flanked by a stretch of three negatively charged residues. In animals, the tyrosine of the HbYX motif can be phosporylated in vivo [120–122]. The extension serves as binding site for other factors and is also thought to help Cdc48 to dock onto the proteasome. The secondary structure elements are shown according to [18]. (B) Structure of the tandem D-domains of Cdc48 (PDB: 3CF1, [43]). Important structure motifs are colored as in (A). (AI 3 mb