A Deubiquitylating Complex Required for Neosynthesis of a Yeast Mitochondrial ATP Synthase Subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis

Versatile Roles of K63-Linked Ubiquitin Chains in Trafficking

Modification by Lys63-linked ubiquitin (UbK63) chains is the second most abundant form of ubiquitylation. In addition to their role in DNA repair or kinase activation, UbK63 chains interfere with multiple steps of intracellular trafficking. UbK63 chains decorate many plasma membrane proteins, providing a signal that is often, but not always, required for their internalization. In yeast, plants, worms and mammals, this same modification appears to be critical for efficient sorting to multivesicular bodies and subsequent lysosomal degradation. UbK63 chains are also one of the modifications involved in various forms of autophagy (mitophagy, xenophagy, or aggrephagy). Here, in the context of trafficking, we report recent structural studies investigating UbK63 chains assembly by various E2/E3 pairs, disassembly by deubiquitylases, and specifically recognition as sorting signals by receptors carrying Ub-binding domains, often acting in tandem. In addition, we address emerging and unanticipated roles of UbK63 chains in various recycling pathways that function by activating nucleators required for actin polymerization, as well as in the transient recruitment of signaling molecules at the plasma or ER membrane. In this review, we describe recent advances that converge to elucidate the mechanisms underlying the wealth of trafficking functions of UbK63 chains

Study of the topogenesis mechanisms of transmembrane transporters in Aspergillus nidulans

Apart from being precursors of proteins, amino acids can support growth of microbial organisms, are involved in osmoregulation in plants and neurotransmission in mammals. Amino acid uptake from the extracellular environment is mediated by specific polytopic transmembrane proteins of the plasma membrane, also known as amino acid transporters. The study of amino acid transporters is so far focused on model microbial systems, such as the ascomycetes Saccharomyces cerevisiae and Aspergillus nidulans, where genetic, molecular and biochemical approaches are more easily applied compared to higher eukaryotes. Fungal amino acid transporters are members of the evolutionary conserved APC (Amino acid Polyamine organoCation) superfamily. In A. nidulans, three transporters have been characterized in a genetic level: the major proline transporter PrnB, the γ-amino butyric transporter GabA and the acidic amino acid transporter AgtA. Limited information is available concerning the post-translational targeting of these proteins to the plasma membrane (topogenesis), the physiological regulation of this process, as well as the cis-regulatory elements and the trans-protein components that coordinate it. During the present PhD thesis, A. nidulans strains expressing functional chimeric AgtA-sGFP transporters have been isolated. In the presence of poor nitrogen sources, such as urea or acidic amino acids, the chimeric molecules AgtA-sGFP are localized in the plasma membrane and the vacuoles of the fungal germlings. Vacuoles are the final destination of these proteins where they become degraded. Addition of a rich nitrogen source, like ammonium, in the growth medium induces the endocytosis of the AgtA-sGFP molecules and their targeting to the vacuoles. Additionally, the shrA gene of A. nidulans has been cloned by in silico analysis and functionally characterized. ShrA is an ER transmembrane protein. The shrA gene expression is constitutive. Deletion of the respective genetic locus causes significant decrease in the ability of A. nidulans to take up proline and aspartate from the growth medium. In the shrAΔ genetic background, the chimeric transporters PrnB-sGFP and AgtA-sGFP are not being properly targeted to the plasma membrane, but remain blocked in intracellular compartments. Thus, ShrA is involved in the post- translational expression of the proline and aspartate transporters in A. nidulans. The shrA cDNA clone also complements the yeast shr3Δ mutation partially. In conclusion, the ShrA protein of A. nidulans is an orthologue of the yeast Shr3p, a molecular chaperon acting at the level of ER exit, specific exclusively for the amino acid transporter family. Further investigation of the genetic or physical interactions in which the ShrA protein is involved may reveal new cis- or trans-factors regulating the topogenesis of amino acid transporters in A. nidulans.Τα αμινοξέα, εκτός από την πρωτεϊνοσύνθεση, εμπλέκονται σε μια σειρά βιολογικών διεργασιών (μικροβιακή αύξηση, οσμωπροστασία στα φυτά, νευροδιαβίβαση στα θηλαστικά). Η πρόσληψη των αμινοξέων από το εξωτερικό περιβάλλον πραγματοποιείται από εξειδικευμένες πολυτοπικές διαμεμβρανικές πρωτεΐνες της πλασματικής μεμβράνης, τους μεταφορείς αμινοξέων. Έως τώρα, η μελέτη των μεταφορέων αμινοξέων έχει επικεντρωθεί σε πρότυπα μικροβιακά συστήματα, όπως οι ασκομύκητες Saccharomyces cerevisiae και Aspergillus nidulans, όπου οι γενετικοί, μοριακοί και βιοχημικοί χειρισμοί καθίστανται ευκολότεροι σε σχέση με τους ανώτερους ευκαρυωτικούς οργανισμούς. Οι μεταφορείς αμινοξέων των μυκήτων είναι μέλη της συντηρημένης σε όλα τα βασίλεια υπεροικογένειας APC (Amino acid/Polyamine/organoCation). Στον A. nidulans τρεις μεταφορείς αμινοξέων έχουν χαρακτηριστεί σε γενετικό επίπεδο: ο κύριος μεταφορέας προλίνης PrnB, ο μεταφορέας γ-αμινοβουτυρικού οξέος GabA και ο μεταφορέας όξινων αμινοξέων AgtA. Ελάχιστα είναι γνωστά όσον αφορά τη μετα-μεταφραστική στόχευση των πρωτεϊνών αυτών στην πλασματική μεμβράνη (τοπογένεση), καθώς και τις περιβαλλοντικές συνθήκες, τα cis-ρυθμιστικά στοιχεία και τους trans-πρωτεϊνικούς παράγοντες που τη ρυθμίζουν. Στα πλαίσια της συγκεκριμένης διδακτορικής διατριβής, απομονώθηκαν στελέχη του Α. nidulans που εκφράζουν λειτουργικούς χιμαιρικούς μεταφορείς AgtA-sGFP. Παρουσία δευτερευουσών πηγών αζώτου, όπως είναι η ουρία ή τα όξινα αμινοξέα, τα χιμαιρικά μόρια AgtA-sGFP εντοπίζονται στην πλασματική μεμβράνη και τα χυμοτόπια των εκβλαστημένων κονιδιοσπορίων. Τα χυμοτόπια αποτελούν θέσεις αποικοδόμησης των μορίων του μεταφορέα στα πλαίσια της φυσιολογικής τους ανακύκλωσης. Η προσθήκη ιόντων αμμωνίου, μίας κύριας πηγής αζώτου, στο θρεπτικό υπόστρωμα επάγει την ενδοκύτωση των μορίων AgtA-sGFP και τη μεταφορά τους στα χυμοτόπια. Επιπλέον, μετά από in silico ανάλυση, κλωνοποιήθηκε και χαρακτηρίστηκε λειτουργικά το γονίδιο shrA του Α. nidulans. Η πρωτεΐνη ShrA είναι διαμεμβρανική και εντοπίζεται στο ενδοπλασματικό δίκτυο του κυττάρου. Η έκφραση του γονιδίου shrA είναι συστατική. Η εξάλειψη του γενετικού τόπου shrA από το γονιδίωμα του Α. nidulans προκαλεί μείωση της ικανότητας πρόσληψης προλίνης και ασπαρτικού οξέος από το θρεπτικό υπόστρωμα. Σε γενετικό υπόβαθρο shrΑΔ, οι χιμαιρικοί μεταφορείς PrnB-sGFP και AgtA-sGFP δεν στοχεύονται στην πλασματική μεμβράνη, αλλά παραμένουν σε ενδοκυτταρικά διαμερίσματα. Συνεπώς, η πρωτεΐνη ShrA εμπλέκεται στη μετα-μεταφραστική έκφραση των μεταφορέων προλίνης και ασπαρτικού οξέος του Α. nidulans. Επιπλέον, ο κλώνος cDNA του γονιδίου shrA συμπληρώνει μερικώς τη μεταλλαγή shr3Δ της ζύμης. Συνεπώς, η πρωτεΐνη ShrA είναι ορθόλογη της πρωτεΐνης Shr3 της ζύμης, η οποία δρα ως μοριακή συνοδός κατά την έξοδο από τη μεμβράνη του ενδοπλασματικού δικτύου για τους μεταφορείς αμινοξέων αποκλειστικά. Η διερεύνηση των φυσικών ή γενετικών αλληλεπιδράσεων στις οποίες εμπλέκεται η πρωτεΐνη ShrA μπορεί να οδηγήσει στην ταυτοποίηση νέων cis- και trans-παραγόντων που ρυθμίζουν την τοπογένεση των μεταφορέων αμινοξέων στον A. nidulans

Versatile Roles of K63-Linked Ubiquitin Chains in Trafficking

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From dysfunctional endoplasmic reticulum-mitochondria coupling to neurodegeneration

International audienceOver the last years, contact sites between the endoplasmic reticulum (ER) and mitochondria have attracted great attention in the study of cell homeostasis and dysfunction, especially in the context of neurodegenerative disorders. This is largely due to the critical involvement of this subcellular compartment in a plethora of vital cellular functions: Ca2+ homeostasis, mitochondrial dynamics, transport, bioenergetics and turnover, ER stress, apoptotic signaling and inflammation. An increasing number of disease-associated proteins have been reported to physically associate with the ER-mitochondria interface, and cause structural and/or functional perturbations of this compartment. In the present review, we summarize current knowledge about the architecture and functions of the ER-mitochondria contact sites, and the consequences of their alteration in different neurodegenerative disorders. Special emphasis is placed on the caveats and difficulties in defining the nature and origin of the highlighted defects in ER-mitochondria communication, and their exact contribution to the neurodegenerative process

The ubiquitin code of yeast permease trafficking.

Yeast permeases, that act as transporters for nutrients including amino acids, nucleobases and metals, provide a powerful model system for dissecting the physiological control of membrane protein trafficking. Modification of these transporters by ubiquitin is known to target them for degradation in the vacuole, the degradation organelle of fungi. Recent studies have uncovered the role of specific adaptors for recruiting the Rsp5 ubiquitin ligase to these proteins. In addition, the role of ubiquitin at different trafficking steps including early endocytosis, sorting into the multivesicular body (MVB) pathway and Golgi-to-endosome transit is now becoming clear. In particular, K63-linked ubiquitin chains now emerge as a specific signal for protein sorting into the MVB pathway. A complete view of the ubiquitin code governing yeast permease trafficking might not be far off.Journal ArticleResearch Support, Non-U.S. Gov'tReviewSCOPUS: re.jinfo:eu-repo/semantics/publishe

Substrate and ubiquitin-dependent trafficking of the yeast siderophore transporter Sit1.

International audienceEukarotic plasma membrane transporters are subjected to a tightly regulated intracellular trafficking. The yeast siderophore transporter Sit1 displays substrate-regulated trafficking. It is targeted to the plasma membrane or to a vacuolar degradative pathway when synthesized in the presence or absence of external substrate, respectively. Sorting of Sit1 to the vacuolar pathway is dependent on the clathrin adapter Gga2, and more specifically on its C-GAT subdomain. Plasma membrane Sit1-GFP undegoes substrate-induced ubiquitylation dependent on the HECT Rsp5 ubiquitin protein ligase. Sit1 is also ubiquitylated in an Rsp5-dependent manner in internal compartments when expressed in the absence of substrate. In several rsp5 mutants including cells deleted for RSP5, Sit1 expressed in the absence of substrate is correctly targeted to the endosomal pathway, but its sorting to multivesicular bodies (MVBs) is impaired. Consequently, it displays endosome to plasma membrane targeting, with kinetics similar to those observed in vps mutants defective for MVB sorting. Plasma membrane Sit1 is modified by Lys63-linked ubiquitin chains. We also show for the first time in yeast that modification by this latter type of ubiquitin chains is required directly or indirectly for efficient MVB sorting, as it is for efficient internalization at the plasma membrane

An ubiquitin-dependent balance between mitofusin turnover and fatty acids desaturation regulates mitochondrial fusion

International audienceMitochondrial integrity relies on homotypic fusion between adjacent outer membranes, which is mediated by large GTPases called mitofusins. The regulation of this process remains nonetheless elusive. Here, we report a crosstalk between the ubiquitin protease Ubp2 and the ubiquitin ligases Mdm30 and Rsp5 that modulates mitochondrial fusion. Ubp2 is an antagonist of Rsp5, which promotes synthesis of the fatty acids desaturase Ole1. We show that Ubp2 also counteracts Mdm30-mediated turnover of the yeast mitofusin Fzo1 and that Mdm30 targets Ubp2 for degradation thereby inducing Rsp5-mediated desaturation of fatty acids. Exogenous desaturated fatty acids inhibit Ubp2 degradation resulting in higher levels of Fzo1 and maintenance of efficient mitochondrial fusion. Our results demonstrate that the Mdm30-Ubp2-Rsp5 crosstalk regulates mitochondrial fusion by coordinating an intricate balance between Fzo1 turnover and the status of fatty acids saturation. This pathway may link outer membrane fusion to lipids homeostasis

Végétalisation du corps lipidique de S. cerevisiae pour l'étude structurale de protéines intégrales et la production de biomasse lipidique

Dans le contexte actuel d’épuisement des ressources fossiles et de protection de l’environnement, la valorisation énergétique des huiles issues de la biomasse et la chimie verte prennent de l’importance. En effet, ces huiles et leurs dérivés biodégradables peuvent venir en remplacement des produits d'origine fossile. Ils sont de plus en plus retrouvés dans les produits de grande consommation (savon, produits d’entretien) ou industriels (solvants, lubrifiants). Deux sources sont envisagées, celles des huiles végétales déjà bien implantées, et celle des huiles produites à partir de microorganismes, actuellement en plein essor. Nos travaux visent à identifier des facteurs influant sur la qualité et la quantité de lipides produits par l’amélioration des plantes ou la transformation génétique de levures. Les lipides de réserve sont stockés dans des granules, les corps lipidiques, puis mobilisés en cas de besoins nutritionnels. Ces structures sont présentes chez les eucaryotes supérieurs (mammifères, plantes) et chez certains unicellulaires (bactéries, levures). Les corps lipidiques sont constitués d’un cœur de lipides neutres, entourés par une monocouche de phospholipides dans laquelle sont insérées de nombreuses protéines. En particulier, il existe des protéines structurales hydrophobes qui stabilisent l'interface entre ces inclusions lipidiques et le milieu aqueux (protéines PAT, apolipoprotéines, oléosines). Les oléosines, de masse comprise entre 15 et 25 kDa, sont les protéines majoritaires du corps lipidique de plantes et recouvrent entièrement sa surface. Grâce à l'expression hétérologue chez S. cerevisiae de deux de ces protéines, l'oléosine AtOle1 et la caléosine AtClo1 de la graine d’Arabidopsis thaliana, nous avons démontré que ces protéines s'insèrent à la surface des corps lipidiques en très grande quantité et favorisent la prolifération de ces structures. Nous avons obtenu une augmentation de + 46.6 % de la teneur en acides gras pour la souche exprimant AtClo1. Le travail se poursuit autour de cette souche modifiée pour le développement de systèmes biologiques performants de production des huiles. Nous utilisons également ces corps lipidiques "végétalisés" pour déterminer le repliement des oléosines par des analyses structurales (SRCD) de ces protéines très hydrophobes dans un environnement naturel afin de développer leur valorisation comme émulsifiant vert par exempl

Versatile role of the yeast ubiquitin ligase Rsp5p in intracellular trafficking.

International audienceThe ubiquitin ligase (E3) Rsp5p is the only member of the Nedd (neural-precursor-cell-expressed, developmentally down-regulated) 4 family of E3s present in yeast. Rsp5p has several proteasome-independent functions in membrane protein trafficking, including a role in the ubiquitination of most plasma membrane proteins, leading to their endocytosis. Rsp5p is also required for the ubiquitination of endosomal proteins, leading to their sorting to the internal vesicles of MVBs (multivesicular bodies). Rsp5p catalyses the attachment of non-conventional ubiquitin chains, linked through ubiquitin Lys-63, to some endocytic and MVB cargoes. This modification appears to be required for efficient sorting, possibly because these chains have a greater affinity for the ubiquitin-binding domains present within endocytic or MVB sorting complexes. The mechanisms involved in the recognition of plasma membrane and MVB substrates by Rsp5p remain unclear. A subset of Rsp5/Nedd4 substrates have a 'PY motif' and are recognized directly by the WW (Trp-Trp) domains of Rsp5p. Most Rsp5p substrates do not carry PY motifs, but some may depend on PY-containing proteins for their ubiquitination by Rsp5p, consistent with the latter's acting as specificity factors or adaptors. As in other ubiquitin-conjugating systems, these adaptors are also Rsp5p substrates and undergo ubiquitin-dependent trafficking. In the present review, we discuss recent examples illustrating the role of Rsp5p in membrane protein trafficking and providing new insights into the regulation of this E3 by adaptor proteins