Publications by Year: 2015

Martzoukou O, Karachaliou M, Yalelis V, Leung J, Byrne B, Amillis S, Diallinas G. Oligomerization of the UapA purine transporter is critical for ER-exit, plasma membrane localization and turnover. J Mol Biol. 2015.Abstract
Central to the process of transmembrane cargo trafficking is the successful folding and exit from the ER through packaging in COPII vesicles. Here, we use the UapA purine transporter of Aspergillus nidulans to investigate the role of cargo oligomerization in membrane trafficking. We show that UapA oligomerizes (at least dimerizes) and that oligomerization persists upon UapA endocytosis and vacuolar sorting. Using a validated BiFC assay, we provide evidence that a UapA oligomerization is associated with ER exit and turnover, as ER-retained mutants, either due to modification of a Tyr-based N-terminal motif or partial misfolding, physically associate, but do not associate properly. Co-expression of ER-retained mutants with wild-type UapA leads to in trans plasma membrane localization of the former, confirming that oligomerization initiates in the ER. Genetic suppression of an N-terminal mutation in the Tyr motif and mutational analysis suggest that transmembrane α-helix 7 affects the oligomerization interface. Our results reveal that transporter oligomerization is essential for membrane trafficking and turnover and is a common theme in fungi and mammalian cells.
Kankipati HN, Rubio-Texeira M, Castermans D, Diallinas G, Thevelein JM. Sul1 and Sul2 Sulfate Transceptors Signal to Protein Kinase A upon Exit of Sulfur Starvation. J Biol Chem. 2015.Abstract
Sulfate is an essential nutrient with pronounced regulatory effects on cellular metabolism and proliferation. Little is known, however, about how sulfate is sensed by cells. Sul1 and Sul2 are sulfate transporters in the yeast Saccharomyces cerevisiae, strongly induced upon sulfur starvation and endocytosed upon addition of sulfate. We reveal Sul1,2-dependent activation of protein kinase A (PKA) targets upon sulfate-induced exit from growth arrest after sulfur starvation. We provide two major arguments in favor of Sul1 and Sul2 acting as transceptors for signaling to PKA. First, the sulfate analogue, D-glucosamine 2-sulfate, acted as a non-transported agonist of signaling by Sul1 and Sul2. Second, mutagenesis to Gln of putative H+-binding residues, Glu427 in Sul1 or Glu443 in Sul2, abolished transport without affecting signaling. Hence, Sul1,2 can function as pure sulfate sensors. Sul1E427Q and Sul2E443Q are also deficient in sulfate-induced endocytosis, which can therefore be uncoupled from signaling. Overall, our data suggest that transceptors can undergo independent conformational changes each responsible for triggering different downstream processes. The Sul1 and Sul2 transceptors are the first identified plasma membrane sensors for extracellular sulfate. High-affinity transporters induced upon starvation for their substrate may generally act as transceptors during exit from starvation.
Krypotou E, Evangelidis T, Bobonis J, Pittis AA, Gabaldón T, Scazzocchio C, Mikros E, Diallinas G. Origin, diversification and substrate specificity in the family of NCS1/FUR transporters. Mol Microbiol. 2015.Abstract
NCS1 proteins are H(+) /Na(+) symporters specific for the uptake of purines, pyrimidines and related metabolites. In this article we study the origin, diversification and substrate specificity of fungal NCS1 transporters. We show that the two fungal NCS1 subfamilies, Fur and Fcy, and plant homologues, originate through independent horizontal transfers from prokaryotes, and that expansion by gene duplication led to the functional diversification of fungal NCS1. We characterized all Fur proteins of the model fungus Aspergillus nidulans and discovered novel functions and specificities. Homology modelling, substrate docking, molecular dynamics and systematic mutational analysis in three Fur transporters with distinct specificities identified residues critical for function and specificity, located within a major substrate binding site, in transmembrane segments TMS1, TMS3, TMS6 and TMS8. Most importantly, we predict and confirm that residues determining substrate specificity are located not only in the major substrate binding site, but also in a putative outward-facing selective gate. Our evolutionary and structure-function analysis contributes in the understanding of the molecular mechanisms underlying the functional diversification of eukaryotic NCS1 transporters, and in particular, forward the concept that selective channel-like gates might contribute to substrate specificity.