Diverse functions of mRNA metabolism factors in stress defense and aging of Caenorhabditis elegans.

Processing bodies (PBs) and stress granules (SGs) are related, cytoplasmic RNA-protein complexes that contribute to post-transcriptional gene regulation in all eukaryotic cells. Both structures contain translationally repressed mRNAs and several proteins involved in silencing, stabilization or degradation of mRNAs, especially under environmental stress. Here, we monitored the dynamic formation of PBs and SGs, in somatic cells of adult worms, using fluorescently tagged protein markers of each complex. Both complexes were accumulated in response to various stress conditions, but distinct modes of SG formation were induced, depending on the insult. We also observed an age-dependent accumulation of PBs but not of SGs. We further showed that direct alterations in PB-related genes can influence aging and normal stress responses, beyond their developmental role. In addition, disruption of SG-related genes had diverse effects on development, fertility, lifespan and stress resistance of worms. Our work therefore underlines the important roles of mRNA metabolism factors in several vital cellular processes and provides insight into their diverse functions in a multicellular organism.

The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans.

The general control nonderepressible 2 (GCN2) kinase is a nutrient-sensing pathway that responds to amino acids deficiency and induces a genetic program to effectively maintain cellular homeostasis. Here we established the conserved role of Caenorhabditis elegans GCN-2 under amino acid limitation as a translation initiation factor 2 (eIF2) kinase. Using a combination of genetic and molecular approaches, we showed that GCN-2 kinase activity plays a central role in survival under nutrient stress and mediates lifespan extension conferred by dietary restriction (DR) or inhibition of the major nutrient-sensing pathway, the target of rapamycin (TOR). We also demonstrated that the GCN-2 and TOR signaling pathways converge on the PHA-4/FoxA transcription factor and its downstream target genes to ensure survival of the whole organism under a multitude of stress conditions, such as nutrient scarcity or environmental stresses. This is one step forward in the understanding of evolutionary conserved mechanisms that confer longevity and healthspan.

Transport-dependent endocytosis and turnover of a uric acid-xanthine permease.

In this work we unmask a novel downregulation mechanism of the uric acid/xanthine transporter UapA, the prototype member of the ubiquitous Nucleobase-Ascorbate Transporter family, directly related to its function. In the presence of substrates, UapA is endocytosed, sorted into the multivesicular body pathway and degraded in vacuoles. Substrate-induced endocytosis, unlike ammonium-induced turnover, is absolutely dependent on UapA activity and several lines of evidence showed that the signal for increased endocytosis is the actual translocation of substrates through the UapA protein. The use of several UapA functional mutants with altered kinetics and specificity has further shown that transport-dependent UapA endocytosis occurs through a mechanism, which senses subtle conformational changes associated with the transport cycle. We also show that distinct mechanisms of UapA endocytosis necessitate ubiquitination of a single Lys residue (K572) by HulA(Rsp5). Finally, we demonstrate that in the presence of substrates, non-functional UapA versions can be endocytosed in trans if expressed in the simultaneous presence of active UapA versions, even if the latter cannot be endocytosed themselves.

Specific interdomain synergy in the UapA transporter determines its unique specificity for uric acid among NAT carriers.

UapA, a uric acid-xanthine permease of Aspergillus nidulans, has been used as a prototype to study structure-function relationships in the ubiquitous nucleobase-ascorbate transporter (NAT) family. Using novel genetic screens, rational mutational design, chimeric NAT molecules, and extensive transport kinetic analyses, we show that dynamic synergy between three distinct domains, transmembrane segment (TMS)1, the TMS8-9 loop, and TMS12, defines the function and specificity of UapA. The TMS8-9 loop includes four residues absolutely essential for substrate binding and transport (Glu356, Asp388, Gln408, and Asn409), whereas TMS1 and TMS12 seem to control, through steric hindrance or electrostatic repulsion, the differential access of purines to the TMS8-9 domain. Thus, UapA specificity is determined directly by the specific interactions of a given substrate with the TMS8-9 loop and indirectly by interactions of this loop with TMS1 and TMS12. We finally show that intramolecular synergy among UapA domains is highly specific and propose that it forms the basis for the evolution of the unique specificity of UapA for uric acid, a property not present in other NAT members.

The Aspergillus nidulans FcyB cytosine-purine scavenger is highly expressed during germination and in reproductive compartments and is downregulated by endocytosis.

We cloned and characterized an Aspergillus nidulans gene, called fcyB, encoding the closest homologue to the yeast Fcy2p/Fcy21p permeases. Deletion of fcyB (DeltafcyB) does not affect growth, development, reproduction or bulk purine uptake, but eliminates the leaky growth on purines of DeltaazgADeltauapCDeltauapA strains, lacking all known purine transporters, and confers resistance to the antifungal 5-fluorocytosine. Kinetic analyses showed FcyB is a low-capacity, high-affinity, cytosine-purine transporter sharing similar molecular interactions for substrate recognition with the yeast Fcy2p/Fcy21p carriers. fcyB transcription is highly activated during germination but drops at low constitutive levels throughout vegetative development. UaY-mediated purine induction of fcyB transcription is only moderate, while ammonium represses transcription through an AreA-dependent mechanism. A strain expressing FcyB-GFP confirms a low protein expression level in the plasma membrane of vegetative mycelia, but reveals an abundant expression in sexual and asexual compartments. FcyB-GFP was also shown to be downregulated by endocytosis in response to ammonia or the presence of cytosine. The expression profile of FcyB supports that its main physiological role is cytosine-purine scavenging.

A novel-type substrate-selectivity filter and ER-exit determinants in the UapA purine transporter.

We present a functional analysis of the last alpha-helical transmembrane segment (TMS12) of UapA, a uric acid-xanthine/H+ symporter in Aspergillus nidulans and member of the nucleobase-ascorbate transporter (NAT) family. First, we performed a systematic mutational analysis of residue F528, located in the middle of TMS12, which was known to be critical for UapA specificity. Substitution of F528 with non-aromatic amino acid residues (Ala, Thr, Ser, Gln, Asn) did not affect significantly the kinetics of UapA for its physiological substrates, but allowed high-capacity transport of several novel purines and pyrimidines. Allele-specific combinations of F528 substitutions with mutations in Q408, a residue involved in purine binding, led to an array of UapA molecules with different kinetic and specificity profiles. We propose that F528 plays the role of a novel-type selectivity filter, which, in conjunction with a distinct purine-binding site, control UapA-mediated substrate translocation. We further studied the role of TMS12 by analysing the effect of its precise deletion and chimeric molecules in which TMS12 was substituted with analogous domains from other NATs. The presence of any of the TMS12 tested was necessary for ER-exit while their specific amino acid composition affected the kinetics of chimeras.

The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway.

UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H+ symporter of Aspergillus nidulans. We have previously presented evidence showing that a highly conserved signature motif ([Q/E/P]408-N-X-G-X-X-X-X-T-[R/K/G])417 is involved in UapA function. Here, we present a systematic mutational analysis of conserved residues in or close to the signature motif of UapA. We show that even the most conservative substitutions of residues Q408, N409 and G411 modify the kinetics and specificity of UapA, without affecting targeting in the plasma membrane. Q408 substitutions show that this residue determines both substrate binding and transport catalysis, possibly via interactions with position N9 of the imidazole ring of purines. Residue N409 is an irreplaceable residue necessary for transport catalysis, but is not involved in substrate binding. Residue G411 determines, indirectly, both the kinetics (K(m), V) and specificity of UapA, probably due to its particular property to confer local flexibility in the binding site of UapA. In silico predictions and a search in structural databases strongly suggest that the first part of the NAT signature motif of UapA (Q(408)NNG(411)) should form a loop, the structure of which is mostly affected by mutations in G411. Finally, substitutions of residues T416 and R417, despite being much better tolerated, can also affect the kinetics or the specificity of UapA. Our results show that the NAT signature motif defines the function of the UapA purine translocation pathway and strongly suggest that this might occur by determining the interactions of UapA with the imidazole part of purines.