The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

International audienceThe DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrom-etry analyses, genetic experiments, sucrose gradients , in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs

Heterologous expression of a plant uracil transporter in yeast: improvement of plasma membrane targeting in mutants of the Rsp5p ubiquitin protein ligase.

Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents. However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 (AtUPS1), previously cloned by functional complementation in yeast. Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane-bound proteins. rsp5 mutants displayed further plasma membrane stabilization of untagged AtUPS1, and improved steady state amounts of tagged versions of AtUPS1. rsp5 cells are thus powerful tools to solve the many problems inherent in heterologous expression of membrane proteins in yeast, including ER retention

Trafficking of Siderophore Transporters in Saccharomyces cerevisiae and Intracellular Fate of Ferrioxamine B Conjugates

We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process

Ubiquitination of ERMES components by the E3 ligase Rsp5 is involved in mitophagy

International audienceMitochondria are dynamic organelles that undergo permanent fission and fusion events. These processes play an essential role in maintaining normal cellular function. In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum-mitochondrial encounter structure (ERMES) is a marker of sites of mitochondrial division, but it is also involved in a plethora of other mitochondrial functions. However, it remains unclear how these different functions are regulated. We show here that Mdm34 and Mdm12, 2 components of ERMES, are ubiquitinated by the E3 ligase Rsp5. This ubiquitination is not involved in mitochondrial dynamics or in the distribution and turnover of ERMES. Nevertheless, the ubiquitination of Mdm34 and Mdm12 was required for efficient mitophagy. We thus report here the first identification of ubiquitinated substrates participating in yeast mitophagy

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

Heterologous expression of a plant uracil transporter in yeast: improvement of plasma membrane targeting in mutants of the Rsp5p ubiquitin protein ligase.

Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents. However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 (AtUPS1), previously cloned by functional complementation in yeast. Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane-bound proteins. rsp5 mutants displayed further plasma membrane stabilization of untagged AtUPS1, and improved steady state amounts of tagged versions of AtUPS1. rsp5 cells are thus powerful tools to solve the many problems inherent in heterologous expression of membrane proteins in yeast, including ER retention

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

Régulation différentielle du trafic des transporteurs de sidérophores de membrane plasmique chez la levure

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The RNA Helicase Ded1 from Yeast Is Associated with the Signal Recognition Particle and Is Regulated by SRP21

The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation