Nitrogen-regulated Ubiquitination of the Gap1 Permease of Saccharomyces cerevisiae

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces rapid inactivation and degradation of the general amino acid permease Gap1 through a process requiring the Npi1/Rsp5 ubiquitin (Ub) ligase. In this study, we show that NH4+ induces endocytosis of Gap1, which is then delivered into the vacuole where it is degraded. This down-regulation is accompanied by increased conversion of Gap1 to ubiquitinated forms. Ubiquitination and subsequent degradation of Gap1 are impaired in the npi1 strain. In this mutant, the amount of Npi1/Rsp5 Ub ligase is reduced >10-fold compared with wild-type cells. The C-terminal tail of Gap1 contains sequences, including a di-leucine motif, which are required for NH4+-induced internalization and degradation of the permease. We show here that mutant Gap1 permeases affected in these sequences still bind Ub. Furthermore, we provide evidence that only a small fraction of Gap1 is modified by Ub after addition of NH4+ to mutants defective in endocytosis.

Caractérisation génétique, phénotypique et formation de biofilm des souches de Candida albicans répondant ou non au farnésol

Candida albicans, le pathogène opportuniste le plus commun, peut subir des transitions morphologiques entre la forme levure et la forme hyphe, jouant un rôle dans la formation de biofilm. Le farnésol, un lipide endogène produit par C. albicans, est une molécule de quorum sensing qui inhibe cette transition morphologique. Certaines souches ne répondent pas au farnésol et nous avons vérifié les hypothèses que : 1) l’isolat clinique SC5314, la souche la mieux caractérisée, est un répondeur au farnésol; 2) la germination, la croissance et la formation de biofilm des non répondeurs diffèrent des répondeurs; 3) l’absence de la réponse au farnésol se manifeste en dehors de conditions de culture précises; 4) le farnésol agit via un récepteur nucléaire qui présente des altérations chez les non répondeurs; 5) la différence de la réponse au farnésol entre les souches s’explique par des variations au niveau transcriptionnel de certains gènes (CHK1, HST7, CPH1, GAP1, RAM2 et DPP3). Les non répondeurs produisent un plus grand nombre d’hyphes, forment 60% plus de biofilm et croissent 50% moins vite que les répondeurs. La souche SC5314 se comporte comme un répondeur. L’absence de la réponse au farnésol se manifeste indépendamment des conditions de culture. Cependant, elle ne s’explique pas par une différence dans le niveau d’expression des gènes proposés, excepté pour DPP3 qui est surexprimé chez le non répondeur ATTC® 36802, suggérant ainsi une surproduction de farnésol chez cette souche. De plus, si le farnésol agit via un récepteur nucléaire, il sera d’un type non décrit précédemment.

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41Δ, sap190Δ, ppm1Δ, rrd1Δ) and U34 modification defects (elp3Δ, kti12Δ, urm1Δ, ncs2Δ) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3Δ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3Δ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3Δ, urm1Δ) enhances nuclear import of and NCR gene activation (MEP2, GAP1) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TORsensitive NCR branch via Gln3 misregulation.

Identification of genes required for Drosophila eye development using a phenotypic enhancer-trap

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (i) the isolation of enhancer-traps with a specific expression pattern, and (ii) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.

Role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes.

We have isolated the NIL1 gene, whose product is an activator of the transcription of nitrogen-regulated genes, by virtue of the homology of its zinc-finger domain to that of the previously identified activator, the product of GLN3. Disruption of the chromosomal NIL1 gene enabled us to compare the effects of Gln3p and of Nil1p on the expression of the nitrogen-regulated genes GLN1, GDH2, and GAP1, coding respectively for glutamine synthetase, NAD-linked glutamate dehydrogenase, and general amino acid permease. Our results show that the nature of GATAAG sequence that serve as the upstream activation sequence elements for these genes determines their abilities to respond to Gln3p and Nil1p. The results further indicate that Gln3p is inactivated by an increase in the intracellular concentration of glutamine and that Nil1p is inactivated by an increase in intracellular glutamate.

Migration and asylum: The movement of people in the Mediterranean Region - Future scenarios and the EU response. Jean Monnet Occasional Paper No. 11/2014

Introduction. In recent years, the global discussion on migration and asylum has evolved from polarization of perspectives and mistrust, to improving partnerships and fostering cooperation between countries and regions. The paradigm has shifted from control and security exclusively to an increased awareness of the ramifications of migration in development and labour markets, the increasing demographic gap1 and the dangers of exclusion faced by migrant workers (regular or irregular). Eastern Europe will suffer the biggest population decline in the coming years, and Nigeria’s population will reach one billion by 2100. In Europe, the work replacement ratio will be two pensioners for one active worker. It has become clear that these facts cannot be ignored and that there is a need for greater convergence of policies (migration/mobility, fundamental rights, and economic growth), with a migrant-centred approach.2. The assumption that Europe will remain a geopolitical and economic hub that attracts immigrants at all skill levels might not hold water in the long run. The evolving demographic and economic changes have made it evident that the competitiveness of the EU (Europe 2020 Strategy) is also at stake, particularly if an adaptable workforce with the necessary skills is not secured in view of shortfalls in skill levels and because of serious labour mismatches. Therefore, it is the right moment to develop more strategic and long-term migration policies that take into account the evolving position of Europe and its neighbours in the world. By the same token, labour market strategies that meet needs and promote integration of regular migrants are still a pending task for the Member States (MS) in terms of the free movement of people, but also in relation with neighbouring and partner countries.