live-cell-imaging-of-early-autophagy-events-omegasomes-and-beyond

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Babraham Institute

Eukaryotic aminoacyl-tRNA synthetases are usually organized into high-molecular-weight complexes, the structure and function of which are poorly understood. We have previously described a yeast complex containing two aminoacyl-tRNA synthetases, methionyl-tRNA synthetase and glutamyl-tRNA synthetase, and one noncatalytic protein, Arc1p, which can stimulate the catalytic efficiency of the two synthetases. To understand the complex assembly mechanism and its relevance to the function of its components, we have generated specific mutations in residues predicted by a recent structural model to be located at the interaction interfaces of the N-terminal domains of all three proteins. Recombinant wild-type or mutant forms of the proteins, as well as the isolated N-terminal domains of the two synthetases, were overexpressed in bacteria, purified and used for complex formation in vitro and for determination of binding affinities using surface plasmon resonance. Moreover, mutant proteins were expressed as PtA or green fluorescent protein fusion polypeptides in yeast strains lacking the endogenous proteins in order to monitor in vivo complex assembly and their subcellular localization. Our results show that the assembly of the Arc1p-synthetase complex is mediated exclusively by the N-terminal domains of the synthetases and that the two enzymes bind to largely independent sites on Arc1p. Analysis of single-amino-acid substitutions identified residues that are directly involved in the formation of the complex in yeast cells and suggested that complex assembly is mediated predominantly by van der Waals and hydrophobic interactions, rather than by electrostatic forces. Furthermore, mutations that abolish the interaction of methionyl-tRNA synthetase with Arc1p cause entry of the enzyme into the nucleus, proving that complex association regulates its subcellular distribution. The relevance of these findings to the evolution and function of the multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases is discussed.

Molecular determinants of the yeast Arc1p-aminoacyl-tRNA synthetase complex assembly.

Eucaryotes aminoacyl-tRNA synth&eacute;tases sont g&eacute;n&eacute;ralement organis&eacute;es dans des complexes de poids mol&eacute;culaire &eacute;lev&eacute;, la structure et la fonction dont on conna&icirc;t mal. Nous avons d&eacute;j&agrave; d&eacute;crit une complexe contenant deux aminoacyl-tRNA synth&eacute;tases, m&eacute;thionyl-tRNA synth&eacute;tase et glutamyl-tRNA synth&eacute;tase et une prot&eacute;ine non catalytique, Arc1p, qui peut stimuler l&#39;efficacit&eacute; catalytique des deux synth&eacute;tases de levure. Pour comprendre le m&eacute;canisme d&#39;assemblage complexe et sa pertinence par rapport &agrave; la fonction de ses composants, nous avons g&eacute;n&eacute;r&eacute; des mutations sp&eacute;cifiques dans les r&eacute;sidus pr&eacute;vues par un mod&egrave;le structurel r&eacute;cent qui sera situ&eacute; &agrave; l&#39;interface d&#39;interaction des domaines N-terminale de tous les trois prot&eacute;ines. Des formes recombinantes sauvage ou mutants des prot&eacute;ines, ainsi que les domaines N-terminaux isol&eacute;s des deux synth&eacute;tases, &eacute;taient surexprim&eacute;s chez les bact&eacute;ries, purifi&eacute;s et utilis&eacute;s pour la formation de complexes in vitro et d&eacute;termination de lier des affinit&eacute;s &agrave; l&#39;aide de la r&eacute;sonance plasmonique de surface. En outre, prot&eacute;ines mutantes ont &eacute;t&eacute; exprim&eacute;es en ATP ou polypeptides de fusion de prot&eacute;ine fluorescente verte en manque les prot&eacute;ines endog&egrave;nes afin de suivre in vivo assemblage complexe et leur localisation subcellulaire des souches de levures. Nos r&eacute;sultats montrent que l&#39;assemblage du complexe Arc1p-synth&eacute;tase est v&eacute;hicul&eacute;e uniquement par les domaines N-terminale des synth&eacute;tases et que les deux enzymes se lient aux sites largement ind&eacute;pendantes sur Arc1p. Analyse des substitutions d&#39;acides amin&eacute;s unique identifi&eacute; les r&eacute;sidus qui sont directement impliqu&eacute;s dans la formation du complexe dans les cellules de levure et a sugg&eacute;r&eacute; que les complexes d&#39;assemblage sont v&eacute;hicul&eacute;e principalement par van der Waals et les interactions hydrophobes et non par des forces &eacute;lectrostatiques. En outre, des mutations qui abolir l&#39;interaction des m&eacute;thionyl-tRNA synth&eacute;tase avec Arc1p causent entr&eacute;e de l&#39;enzyme dans le noyau, prouvant que l&#39;association complexe r&eacute;gule sa distribution subcellulaire. On discute de la pertinence de ces d&eacute;couvertes &agrave; l&#39;&eacute;volution et la fonction des complexes multienzymatiques des eucaryotes aminoacyl-tRNA synth&eacute;tases.

D&eacute;terminants mol&eacute;culaires de la levure assemblage complexe de Arc1p-aminoacyl-ARNt synth&eacute;tase.

Sintetasi amminoacil-tRNA eucariotici sono solitamente organizzati in complessi ad alto peso molecolare, la struttura e la funzione di cui sono capiti male. In precedenza abbiamo descritto un lievito complesso contenente due sintetasi amminoacil-tRNA, methionyl-tRNA sintetasi e glutammil-tRNA sintetasi e una proteina noncatalytic, Arc1p, che pu&ograve; stimolare l&#39;efficienza catalitica dei due sintetasi. Per comprendere il meccanismo complesso assemblaggio e la sua rilevanza alla funzione dei suoi componenti, noi abbiamo generato specifiche mutazioni in residui prevedere da un recente modello strutturale per essere collocato presso le interfacce di interazione dei domini N-terminale di tutte e tre le proteine. Ricombinante forme wild-type o mutanti delle proteine, cos&igrave; come i domini N-terminale isolati della due sintetasi, erano overexpressed in batteri, purificati e utilizzati per la formazione dei complessi in vitro e per la determinazione dell&#39;associazione affinit&agrave; utilizzando la risonanza plasmonica di superficie. Inoltre, proteine mutanti sono stati espressi come PtA o polipeptidi fusione proteina fluorescente verde in ceppi di lievito mancano le proteine endogene al fine di monitorare in vivo assieme complesso e loro localizzazione subcellulare. I nostri risultati mostrano che l&#39;assemblaggio del complesso Arc1p-sintetasi &egrave; mediata esclusivamente per i domini N-terminale della sintetasi e che i due enzimi si legano ai siti in gran parte indipendenti su Arc1p. Analisi del singolo-amminoacido sostituzioni identificati residui che sono direttamente coinvolti nella formazione del complesso in cellule di lievito e suggeriti che insieme complesso &egrave; mediata prevalentemente da interazioni idrofobiche e forze di van der Waals, piuttosto che di forze elettrostatiche. Inoltre, mutazioni che abolire l&#39;interazione di methionyl-tRNA sintetasi con Arc1p causano entrata dell&#39;enzima nel nucleo, dimostrando che associazione complessa disciplina relativa distribuzione subcellulare. La rilevanza di queste scoperte per l&#39;evoluzione e la funzione dei complessi multienzyme della sintetasi amminoacil-tRNA eucariotici &egrave; discusso.

Determinanti molecolari del lievito insieme complesso Arc1p-amminoacil-tRNA sintetasi.

The catalytic core of methionyl-tRNA synthetase (MetRS) is conserved among all life kingdoms but, depending on species origin, is often linked to non-catalytic domains appended to its N- or C-terminus. These domains usually contribute to protein-protein or protein-tRNA interactions but their exact biological role and evolutionary purpose is poorly understood. Yeast MetRS contains an N-terminal appendix that mediates its interaction with the N-terminal part of Arc1p. Association with Arc1p controls the subcellular distribution of MetRS. Furthermore, the C-terminal part of Arc1p harbors a conserved tRNA-binding domain (TRBD) required for the Arc1p-dependent stimulation of the catalytic activity of MetRS. The same TRBD is found directly fused to catalytic domains of plant and nematode MetRS as well as human tyrosyl-tRNA synthetase. To investigate the purpose of Arc1p-MetRS complex formation in yeast, we tested the ability of TRBD to assist the function of MetRS independently of Arc1p. We attached the TRBD directly to the C-terminus of the MetRS catalytic core (MC) by constructing the chimeric protein MC-TRBD. The effect of MC-TRBD expression on yeast cell growth as well as its localization and in vitro aminoacylation activity were analyzed and compared to that of MC alone or wild-type MetRS, both in the absence or presence of Arc1p. We show that MC-TRBD exhibits improved enzymatic activity and can effectively substitute the MetRS-Arc1p binary complex in vivo. Moreover, MC-TRBD, being exclusively cytoplasmic, also mimics the MetRS-Arc1p complex in terms of subcellular localization. Our results suggest that the sole role of the N-terminal appended domain of yeast MetRS is to mediate the indirect association with the TRBD, which, nevertheless, can also function effectively in vivo when directly fused to the catalytic MetRS core.

Incorporation of the Arc1p tRNA-binding domain to the catalytic core of MetRS can functionally replace the yeast Arc1p-MetRS complex.

Le noyau catalytique de m&eacute;thionyl-tRNA synth&eacute;tase (TEMI) est conserv&eacute; parmi tous les royaumes de la vie mais, selon l&#39;origine des esp&egrave;ces, est souvent li&eacute; aux domaines non catalytiques annex&eacute;es &agrave; son N - ou C-terminus. Ces domaines contribuent g&eacute;n&eacute;ralement &agrave; des interactions prot&eacute;ine-prot&eacute;ine ou prot&eacute;ine-tRNA mais leur r&ocirc;le biologique exact et but &eacute;volutionnaire est mal comprise. TEMI de levure contient un appendice N-terminal qui intervient dans son interaction avec la partie N-terminale de Arc1p. Liaison avec Arc1p contr&ocirc;le la distribution subcellulaire des TEMI. En outre, la partie C-terminale de Arc1p abrite un domaine de liaison de tRNA conserv&eacute; (TRBD) requis pour la stimulation de Arc1p d&eacute;pendant de l&#39;activit&eacute; catalytique des TEMI. Le m&ecirc;me TRBD se trouve directement fusionn&eacute;e &agrave; des domaines catalytiques de plante et n&eacute;matodes TEMI mais aussi humaine tyrosyl-tRNA synth&eacute;tase. Afin d&#39;&eacute;tudier le but de la formation d&#39;un complexe Arc1p-TEMI chez la levure, nous avons test&eacute; la capacit&eacute; des TRBD &agrave; aider la fonction des TEMI ind&eacute;pendamment de Arc1p. Nous le TRBD directement attach&eacute; &agrave; l&#39;extr&eacute;mit&eacute; C-terminale du noyau catalytique TEMI (MC) en construisant la prot&eacute;ine chim&eacute;rique MC-TRBD. L&#39;effet de l&#39;expression de MC-TRBD sur la croissance de cellules de levure ainsi que sa localisation et l&#39;activit&eacute; de l&#39;aminoacylation in vitro ont &eacute;t&eacute; analys&eacute;s et compar&eacute;e &agrave; celle des MC TEMI seul ou sauvage, aussi bien en l&#39;absence ou la pr&eacute;sence de Arc1p. Nous montrons que les expositions de MC-TRBD am&eacute;lior&eacute; l&#39;activit&eacute; enzymatique et peut remplacer efficacement le complexe binaire TEMI-Arc1p in vivo. En outre, MC-TRBD, &eacute;tant exclusivement cytoplasmique, imite aussi le complexe TEMI-Arc1p en termes de localisation subcellulaire. Nos r&eacute;sultats sugg&egrave;rent que le r&ocirc;le unique du domaine N-terminal ajout&eacute; de levure TEMI est &agrave; la m&eacute;diation de l&#39;association indirecte avec le TRBD, qui, n&eacute;anmoins, peut &eacute;galement fonctionner efficacement in vivo lorsque fusionn&eacute;es directement au noyau TEMI catalytique.

Incorporation de la domaine de liaison ARNt Arc1p au noyau catalytique des TEMI peut remplacer fonctionnellement le complexe de la levure Arc1p-TEMI.

Il nucleo catalitico di methionyl-tRNA sintetasi (metri) &egrave; conservato tra tutti i regni della vita ma, a seconda dell&#39;origine della specie, &egrave; spesso legato a domini non catalitiche accodati a N - o C-capolinea. Questi domini di solito contribuiscono alle interazioni della proteina-proteina o proteina-tRNA ma il loro esatto ruolo biologico e scopo evolutivo &egrave; capito male. Lievito metri contiene un&#39;appendice N-terminale che media l&#39;interazione con la parte N-terminale del Arc1p. Associazione con Arc1p controlla la distribuzione sottocellulare di metri. Inoltre, la parte C-terminale del Arc1p porti un dominio di tRNA-associazione conservato (TRBD) necessario per la stimolazione Arc1p-dipendente dell&#39;attivit&agrave; catalitica di metri. La stessa TRBD &egrave; trovato direttamente fusa a domini catalitici di pianta e nematodi metri nonch&eacute; umano tyrosyl-tRNA sintetasi. Per indagare lo scopo della formazione dei complessi Arc1p-metri in lievito, abbiamo testato la capacit&agrave; di assistere la funzione di metri indipendentemente da Arc1p TRBD. Abbiamo attaccato la TRBD direttamente al C-terminale del nucleo catalitico metri (MC) costruendo la proteina chimerica TRBD MC. L&#39;effetto dell&#39;espressione MC-TRBD sulla crescita delle cellule di lievito cos&igrave; come la sua localizzazione e aminoacylation in vitro attivit&agrave; sono stati analizzati e rispetto a quello di MC metri da solo o wild-type, sia nell&#39;assenza o la presenza di Arc1p. Ci mostrano che esposizioni MC-TRBD migliorato l&#39;attivit&agrave; enzimatica e pu&ograve; sostituire efficacemente il complesso binario metri-Arc1p in vivo. Inoltre, MC-TRBD, essendo esclusivamente citoplasmatico, imita anche il complesso di Arc1p metri in termini di localizzazione subcellulare. I nostri risultati suggeriscono che l&#39;unico ruolo del dominio N-terminale aggiunto del lievito metri &egrave; di mediare l&#39;associazione indiretta con TRBD, che, tuttavia, pu&ograve; anche funzionare efficacemente in vivo quando direttamente fusa al nucleo catalitico metri.

Incorporazione del dominio Arc1p tRNA-associazione per il nucleo catalitico di metri pu&ograve; sostituire funzionalmente il complesso di lievito Arc1p-metri.

In seguito l&#39;intricata architettura della cellula eucariota, sintesi proteica comporta la formazione di molti complessivi macromolecolari, alcuni dei quali sono composti da enzimi tRNA-aminoacylation. Interazioni della proteina-proteina e proteina-tRNA in questi complessi possono essere facilitate da non catalitica tRNA-binding proteins. Questa recensione si concentra sulla dissezione delle propriet&agrave; molecolari, strutturali e funzionali di una particolare famiglia di tali proteine: lievito Arc1p e suoi omologhi in procarioti ed eucarioti superiori. Essi rappresentano i paradigmi delle strategie impiegate per l&#39;organizzazione delle nanostrutture dinamica e sofisticata supporting organization cellulare spazio-temporale.

Costruzione di Arche per tRNA: struttura e funzione della famiglia Arc1p di non catalitica tRNA-binding proteins.

Following the intricate architecture of the eukaryotic cell, protein synthesis involves formation of many macromolecular assemblies, some of which are composed by tRNA-aminoacylation enzymes. Protein-protein and protein-tRNA interactions in these complexes can be facilitated by non-catalytic tRNA-binding proteins. This review focuses on the dissection of the molecular, structural and functional properties of a particular family of such proteins: yeast Arc1p and its homologues in prokaryotes and higher eukaryotes. They represent paradigms of the strategies employed for the organization of sophisticated and dynamic nanostructures supporting spatio-temporal cellular organization.

Building arks for tRNA: structure and function of the Arc1p family of non-catalytic tRNA-binding proteins.

Suite &agrave; l&#39;architecture complexe de la cellule eucaryote, synth&egrave;se prot&eacute;ique implique la formation de nombreux assemblages macromol&eacute;culaires, dont certains ont &eacute;t&eacute; compos&eacute;es par ARNt-aminoacylation enzymes. Des interactions prot&eacute;ine-prot&eacute;ine et prot&eacute;ine-tRNA dans ces complexes peuvent &ecirc;tre facilit&eacute;es par des prot&eacute;ines liant le tRNA non catalytiques. Cet examen se concentre sur la dissection des propri&eacute;t&eacute;s mol&eacute;culaires, structurales et fonctionnelles d&#39;une famille particuli&egrave;re de ces prot&eacute;ines : levure Arc1p et ses homologues chez les procaryotes et les eucaryotes sup&eacute;rieurs. Ils repr&eacute;sentent des paradigmes des strat&eacute;gies employ&eacute;es pour l&#39;organisation de nanostructures dynamique et sophistiqu&eacute;, soutenant une organisation cellulaire spatio-temporelle.

Construction d&#39;arches pour ARNt : structure et fonction de la famille de Arc1p des prot&eacute;ines non catalytiques ARNt.

Regulation of membrane lipid composition is crucial for many aspects of cell growth and development. Lipins, a novel family of phosphatidate (PA) phosphatases that generate diacylglycerol (DAG) from PA, are emerging as essential regulators of fat metabolism, adipogenesis, and organelle biogenesis. The mechanisms that govern lipin translocation onto membranes are largely unknown. Here we show that recruitment of the yeast lipin (Pah1p) is regulated by PA levels onto the nuclear/endoplasmic reticulum (ER) membrane. Recruitment requires the transmembrane protein phosphatase complex Nem1p-Spo7p. Once dephosphorylated, Pah1p can bind to the nuclear/ER membrane independently of Nem1p-Spo7p via a short amino-terminal amphipathic helix. Dephosphorylation enhances the activity of Pah1p, both in vitro and in vivo, but only in the presence of a functional helix. The helix is required for both phospholipid and triacylglycerol biosynthesis. Our data suggest that dephosphorylation of Pah1p by the Nem1p-Spo7p complex enables the amphipathic helix to anchor Pah1p onto the nuclear/ER membrane allowing the production of DAG for lipid biosynthesis.

A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase.

R&eacute;gulation de la composition lipidique de la membrane est cruciale pour de nombreux aspects de la croissance cellulaire et du d&eacute;veloppement. Lipins, une nouvelle famille de phosphatidate phosphatases (PA) qui g&eacute;n&egrave;rent le diacylglyc&eacute;rol (DAG) de PA, se pr&eacute;sentent comme des r&eacute;gulateurs essentiels de graisse biogen&egrave;se organite, le m&eacute;tabolisme et l&#39;adipogen&egrave;se. Les m&eacute;canismes qui r&eacute;gissent la translocation lipin sur des membranes sont en grande partie inconnus. Nous d&eacute;montrons que le recrutement de la levure lipin (Pah1p) est r&eacute;gul&eacute;e par niveaux de PA sur la membrane du r&eacute;ticulum endoplasmique/nucl&eacute;aire (ER). Recrutement exige la phosphatase de la prot&eacute;ine de transmembrane complexes Nem1p-Spo7p. Une fois d&eacute;phosphoryl&eacute;, Pah1p pouvez cr&eacute;er une liaison &agrave; la membrane nucl&eacute;aire/ER ind&eacute;pendamment de Nem1p-Spo7p via un court amino-terminale amphipathic helix. D&eacute;phosphorylation augmente l&#39;activit&eacute; de Pah1p, deux &eacute;tudes in vitro et in vivo, mais seulement en pr&eacute;sence d&#39;une h&eacute;lice fonctionnelle. L&#39;h&eacute;lice est n&eacute;cessaire pour la biosynth&egrave;se des phospholipides et des triacylglyc&eacute;rols. Nos donn&eacute;es sugg&egrave;rent que d&eacute;phosphorylation de Pah1p par le complex permet de Nem1p-Spo7p l&#39;h&eacute;lice amphipathique ancre Pah1p sur la membrane nucl&eacute;aire/ER, permettant la fabrication de DAG pour la biosynth&egrave;se des lipides.

Une h&eacute;lice amphipathique r&eacute;glement&eacute; phosphorylation contr&ocirc;le la translocation de la membrane et la fonction de la phosphatidate phosphatase de la levure.

Regolamento della composizione lipidica di membrana &egrave; cruciale per molti aspetti della crescita cellulare e lo sviluppo. Lipins, una famiglia romanzo di phosphatidate fosfatasi (PA) che generano diacilglicerolo (DAG) da PA, stanno emergendo come regolatori essenziali della biogenesi dell&#39;organello, adipogenesi e metabolismo grasso. I meccanismi che governano la traslocazione di Dario sulle membrane sono in gran parte sconosciuti. Qui vi mostriamo che assunzione del lievito Dario (Pah1p) &egrave; regolato dai livelli di PA sulla membrana nucleare/endoplasmic endoplasmatico (ER). Reclutamento richiede la fosfatasi della proteina del transmembrane complessi Nem1p-Spo7p. Una volta defosforilata, Pah1p pu&ograve; legarsi alla membrana nucleare/ER indipendentemente dal Nem1p-Spo7p tramite un&#39;elica amphipathic amminico-terminale corto. Defosforilazione migliora l&#39;attivit&agrave; di Pah1p, entrambi in vitro e in vivo, ma solo in presenza di un&#39;elica funzionale. L&#39;elica &egrave; necessaria per la biosintesi di fosfolipidi sia triacilglicerolo. I nostri dati suggeriscono che dephosphorylation di Pah1p dalla Nem1p-Spo7p complessa consente l&#39;elica amphipathic ancoraggio Pah1p sulla membrana nucleare/ER consentendo la produzione di DAG per la biosintesi dei lipidi.

Un&#39;elica amphipathic regolato da fosforilazione controlla la traslocazione di membrana e funzione della fosfatasi phosphatidate lievito.

The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes. PAP is phosphorylated at multiple Ser and Thr residues and is dephosphorylated for in vivo function by the Nem1p-Spo7p protein phosphatase complex localized in the nuclear/endoplasmic reticulum membrane. In this work, we characterized seven previously identified phosphorylation sites of PAP that are within the Ser/Thr-Pro motif. When expressed on a low copy plasmid, wild type PAP could not complement the pah1Î” mutant in the absence of the Nem1p-Spo7p complex. However, phosphorylation-deficient PAP (PAP-7A) containing alanine substitutions for the seven phosphorylation sites bypassed the requirement of the phosphatase complex and complemented the pah1Î” nem1Î” mutant phenotypes, such as temperature sensitivity, nuclear/endoplasmic reticulum membrane expansion, decreased triacylglycerol synthesis, and derepression of INO1 expression. Subcellular fractionation coupled with immunoblot analysis showed that PAP-7A was highly enriched in the membrane fraction. In fluorescence spectroscopy analysis, the PAP-7A showed tighter association with phospholipid vesicles than wild type PAP. Using site-directed mutagenesis of PAP, we identified Ser(602), Thr(723), and Ser(744), which belong to the seven phosphorylation sites, as the sites phosphorylated by the CDC28 (CDK1)-encoded cyclin-dependent kinase. Compared with the dephosphorylation mimic of the seven phosphorylation sites, alanine substitution for Ser(602), Thr(723), and/or Ser(744) had a partial effect on circumventing the requirement for the Nem1p-Spo7p complex.

Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: identification of SER(602), THR(723), AND SER(744) as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.

La Saccharomyces cerevisiae PAH1 cod&eacute; phosphatidate phosphatase (PAP) catalyse l&#39;avant-derni&egrave;re &eacute;tape dans la synth&egrave;se des triacylglyc&eacute;rols et joue un r&ocirc;le dans la r&eacute;gulation transcriptionnelle des phospholipides des g&egrave;nes de synth&egrave;se. PAP est phosphoryl&eacute; &agrave; plusieurs r&eacute;sidus Ser et Thr et est d&eacute;phosphoryl&eacute;e pour fonction in vivo par la Nem1p-Spo7p prot&eacute;ine phosphatase complexe localis&eacute;e dans la membrane du r&eacute;ticulum endoplasmique/nucl&eacute;aire. Dans ce travail, nous avons caract&eacute;ris&eacute; sept sites de phosphorylation pr&eacute;c&eacute;demment identifi&eacute;s de PAP qui se situent dans le motif de Ser/Thr-Pro. Exprim&eacute; sur un plasmide de faibles copies, wild type PAP ne pourraient pas compl&eacute;ter le mutant pah1&Delta; en l&#39;absence du complexe Nem1p-Spo7p. Cependant, phosphorylation d&eacute;ficients en PAP (PAP-7 a) contenant alanine substitutions pour les sites de sept phosphorylation contourn&eacute; l&#39;exigence de la phosphatase complexe et compl&egrave;tent les ph&eacute;notypes mutants de pah1&Delta; nem1&Delta;, comme sensibilit&eacute; &agrave; la temp&eacute;rature, expansion membrane du r&eacute;ticulum endoplasmique/nucl&eacute;aire, la synth&egrave;se des triacylglyc&eacute;rols diminu&eacute;e et d&eacute;r&eacute;pression d&#39;expression INO1. Fractionnement subcellulaire coupl&eacute; &agrave; l&#39;analyse par immunotransfert ont montr&eacute; que le PAP-7 a a &eacute;t&eacute; hautement enrichi dans la fraction de membrane. Dans l&#39;analyse de la spectroscopie de fluorescence, le PAP-7 a a montr&eacute; une association plus serr&eacute;e avec v&eacute;sicules phospholipidiques que type sauvage PAP. &Agrave; l&#39;aide de mutag&eacute;n&egrave;se dirig&eacute;e du PAP, nous avons identifi&eacute; Ser(602), Thr(723) et Ser(744), qui appartiennent &agrave; des sept sites de phosphorylation, comme les sites phosphoryl&eacute;s par la CDC28 (CDK1)-cod&eacute; de kinase cycline-d&eacute;pendante. En comparaison avec l&#39;imitateur de d&eacute;phosphorylation de sept sites de phosphorylation, substitution alanine pour Ser(602), Thr(723), et/ou Ser(744) eu un effet partiel sur contourner l&#39;exigence pour le complexe Nem1p-Spo7p.

La phosphorylation de la phosphatidate phosphatase r&eacute;glemente son association de membrane et les fonctions physiologiques chez Saccharomyces cerevisiae : identification des SER(602), THR(723), et SER(744) que les sites phosphoryl&eacute;s par la CDC28 (CDK1)-cod&eacute; de kinase cycline-d&eacute;pendante.

Il Saccharomyces cerevisiae con codifica PAH1 phosphatidate fosfatasi (PAP) catalizza la seguente il penultimo passo nella sintesi di triacilglicerolo e svolge un ruolo nella regolazione trascrizionale del fosfolipide geni di sintesi. PAP &egrave; fosforilata a pi&ugrave; residui Ser e Thr ed &egrave; defosforilata per funzione in vivo con il complessa di fosfatasi di proteina di Nem1p-Spo7p localizzata nella membrana del reticolo nucleare/endoplasmic. In questo lavoro, abbiamo caratterizzato sette siti precedentemente identificati fosforilazione di PAP che sono all&#39;interno del motivo di Ser/Thr-Pro. Quando espresse su un plasmide copia basso, wild type PAP non potrebbe integrare il mutante pah1&Delta; in assenza del complesso Nem1p-Spo7p. Tuttavia, la fosforilazione-carenti PAP (PAP-7A) contenenti alanina sostituzioni per i siti di sette fosforilazione bypassato il requisito del complesso di fosfatasi e completato fenotipi mutanti di pah1&Delta; nem1&Delta;, come la sensibilit&agrave; alla temperatura, espansione di membrana del reticolo nucleare/endoplasmic, triacilglicerolo diminuita sintesi e derepression di INO1 espressione. Frazionamento subcellulare accoppiato con immunoblot analisi ha mostrato che PAP-7A era altamente arricchito nella frazione di membrana. Nell&#39;analisi di spettroscopia di fluorescenza, il PAP-7A ha mostrato pi&ugrave; stretta associazione con vescicole di fosfolipidi di wild type PAP. Mediante mutagenesi luogo-diretta di PAP, abbiamo identificato Ser(602), Thr(723) e Ser(744), che appartengono ai sette siti di fosforilazione, come i siti fosforilati dal CDC28 (CDK1)-codificato chinasi ciclina-dipendente. Confrontato con il mimic defosforilazione di sette siti di fosforilazione, sostituzione dell&#39;alanina per Ser(602), Thr(723) o Ser(744) ha avuto un effetto parziale su eludere il requisito per il complesso Nem1p-Spo7p.

La fosforilazione di phosphatidate fosfatasi regola la sua associazione di membrana e le funzioni fisiologiche in Saccharomyces cerevisiae: identificazione dei SER(602), THR(723), e SER(744) come i siti fosforilato da CDC28 (CDK1)-codificato chinasi ciclina-dipendente.

Lipins are evolutionarily conserved phosphatidate phosphatases that perform key functions in phospholipid, triglyceride, and membrane biogenesis. Translocation of lipins on membranes requires their dephosphorylation by the Nem1p-Spo7p transmembrane phosphatase complex through a poorly understood mechanism. Here we identify the carboxy-terminal acidic tail of the yeast lipin Pah1p as an important regulator of this step. Deletion or mutations of the tail disrupt binding of Pah1p to the Nem1p-Spo7p complex and Pah1p membrane translocation. Overexpression of Nem1p-Spo7p drives the recruitment of Pah1p in the vicinity of lipid droplets in an acidic tail-dependent manner and induces lipid droplet biogenesis. Genetic analysis shows that the acidic tail is essential for the Nem1p-Spo7p-dependent activation of Pah1p but not for the function of Pah1p itself once it is dephosphorylated. Loss of the tail disrupts nuclear structure, INO1 gene expression, and triglyceride synthesis. Similar acidic sequences are present in the carboxy-terminal ends of all yeast lipin orthologues. We propose that acidic tail-dependent binding and dephosphorylation of Pah1p by the Nem1p-Spo7p complex is an important determinant of its function in lipid and membrane biogenesis.

Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p.

Lipins sont des phosphatases phosphatidate &eacute;volutivement conserv&eacute;s qui ex&eacute;cutent des fonctions cl&eacute;es dans la biogen&egrave;se des phospholipides, de triglyc&eacute;rides et de membrane. Translocation de lipins sur les membranes exige leur d&eacute;phosphorylation de la phosphatase de transmembrane de Nem1p-Spo7p complexe gr&acirc;ce &agrave; un m&eacute;canisme mal compris. Ici, nous identifions la queue acide carboxy-terminale de la levure lipin Pah1p comme un r&eacute;gulateur important de cette &eacute;tape. Suppression ou des mutations de la queue perturbent la liaison de Pah1p &agrave; le Nem1p-Spo7p complexe et la translocation de membrane Pah1p. La surexpression de la Nem1p-Spo7p conduit au recrutement de Pah1p dans les environs de gouttelettes lipidiques d&#39;une fa&ccedil;on d&eacute;pendante de queue acide et induit la biogen&egrave;se des gouttelettes lipidiques. L&#39;analyse g&eacute;n&eacute;tique montre que la queue acide est indispensable pour l&#39;activation de Nem1p-Spo7p-d&eacute;pendante de Pah1p mais pas pour la fonction de Pah1p lui-m&ecirc;me une fois c&#39;est d&eacute;phosphoryl&eacute;e. Perte de la queue perturbe la structure nucl&eacute;aire, l&#39;expression des g&egrave;nes INO1 et synth&egrave;se des triglyc&eacute;rides. S&eacute;quences d&#39;acides semblables sont pr&eacute;sents dans l&#39;extr&eacute;mit&eacute; carboxy-terminal de toutes les levures lipin orthologues. Nous proposons que les acide fixation queue-d&eacute;pendante et d&eacute;phosphorylation de Pah1p par le complexe Nem1p-Spo7p est un facteur d&eacute;terminant de sa fonction dans la biogen&egrave;se des lipides et de la membrane.

Article	Year
Molecular determinants of the yeast Arc1p-aminoacyl-tRNA synthetase complex assembly. Journal of molecular biology\| PubMed ID: 17976650	2007
Incorporation of the Arc1p tRNA-binding domain to the catalytic core of MetRS can functionally replace the yeast Arc1p-MetRS complex. Journal of molecular biology\| PubMed ID: 18598703	2008
Building arks for tRNA: structure and function of the Arc1p family of non-catalytic tRNA-binding proteins. FEBS letters\| PubMed ID: 20728442	2010
A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20876142	2010
Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: identification of SER(602), THR(723), AND SER(744) as... The Journal of biological chemistry\| PubMed ID: 21081492	2011
Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p. Molecular biology of the cell\| PubMed ID: 23657815	2013

Eleftherios Karanasios

Biography

JoVE Journal Publications

Other Publications