Yeast nutrient transceptors provide novel insight in the functionality of membrane transporters (original) (raw)
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BioEssays, 2011
Signaling from transporters provokes re-evaluation of complex trafficking and regulatory controls Endocytic internalization and intracellular trafficking of nutrient transceptors may, at least in part, be governed by their signaling function Johan Kriel 1)2)y , Steven Haesendonckx 1)2)y , Marta Rubio-Texeira 1)2) , Griet Van Zeebroeck 1)2) and Johan M. Thevelein 1)2)Ã When cells are starved of their substrate, many nutrient transporters are induced. These undergo rapid endocytosis and redirection of their intracellular trafficking when their substrate becomes available again. The discovery that some of these transporters also act as receptors, or transceptors, suggests that at least part of the sophisticated controls governing the trafficking of these proteins has to do with their signaling function rather than with control of transport. In yeast, the general amino acid permease Gap1 mediates signaling to the protein kinase A pathway. Its endocytic internalization and intracellular trafficking are subject to amino acid control. Other nutrient transceptors controlling this signal transduction pathway appear to be subject to similar trafficking regulation. Transporters with complex regulatory control have also been suggested to function as transceptors in other organisms. Hence, precise regulation of intracellular trafficking in nutrient transporters may be related to the need for tight control of nutrient-induced signaling.
Transport and signaling via the amino acid binding site of the yeast Gap1 amino acid transceptor
Nature Chemical Biology, 2009
Transporter-related nutrient sensors, called transceptors, mediate nutrient activation of signaling pathways through the plasma membrane. The mechanism of action of transporting and nontransporting transceptors is unknown. We have screened 319 amino acid analogs to identify compounds that act on Gap1, a transporting amino acid transceptor in yeast that triggers activation of the protein kinase A pathway. We identified competitive and noncompetitive inhibitors of transport, either with or without agonist action for signaling, including nontransported agonists. Using substituted cysteine accessibility method (SCAM) analysis, we identified Ser388 and Val389 as being exposed into the amino acid binding site, and we show that agonist action for signaling uses the same binding site as used for transport. Our results provide the first insight, to our knowledge, into the mechanism of action of transceptors. They indicate that signaling requires a ligand-induced specific conformational change that may be part of but does not require the complete transport cycle.
Molecular Microbiology, 2014
The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. L-lysine, L-histidine and L-tryptophan are transported by Gap1 but do not trigger signalling. Unlike Lhistidine, L-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and D-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The nonsignalling agonist, non-transported competitive inhibitor of Gap1 transport, L-Asp-γ-L-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of L-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1 Y395C by ubiquitinationand endocytosis-deficient Gap1 K9R,K16R. Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.
Amino acid transceptors: gate keepers of nutrient exchange and regulators of nutrient signaling
AJP: Endocrinology and Metabolism, 2009
Amino acid transporters at the surface of cells are in an ideal location to relay nutritional information, as well as nutrients themselves, to the cell interior. These transporters are able to modulate signaling downstream of intracellular amino acid receptors by regulating intracellular amino acid concentrations through processes of coupled transport. The concept of dual-function amino acid transporter/receptor (or “transceptor”) proteins is well established in primitive eukaryotes such as yeast, where detection of extracellular amino acid deficiency leads to upregulation of proteins involved in biosynthesis and transport of the deficient amino acid(s). The evolution of the “extracellular milieu” and nutrient-regulated endocrine controls in higher eukaryotes, alongside their frequent inability to synthesize all proteinaceous amino acids (and, hence, the requirement for indispensable amino acids in their diet), appears to have lessened the priority of extracellular amino acid sensin...
Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p
The Journal of Cell Biology, 2006
Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter- like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino ...
Endocytosis of nutrient transporters in fungi: The ART of connecting signaling and trafficking
Computational and Structural Biotechnology Journal, 2021
Plasma membrane transporters play pivotal roles in the import of nutrients, including sugars, amino acids, nucleobases, carboxylic acids, and metal ions, that surround fungal cells. The selective removal of these transporters by endocytosis is one of the most important regulatory mechanisms that ensures a rapid adaptation of cells to the changing environment (e.g., nutrient fluctuations or different stresses). At the heart of this mechanism lies a network of proteins that includes the arrestin-related trafficking adaptors (ARTs) which link the ubiquitin ligase Rsp5 to nutrient transporters and endocytic factors. Transporter conformational changes, as well as dynamic interactions between its cytosolic termini/loops and with lipids of the plasma membrane, are also critical during the endocytic process. Here, we review the current knowledge and recent findings on the molecular mechanisms involved in nutrient transporter endocytosis, both in the budding yeast Saccharomyces cerevisiae and in some species of the filamentous fungus Aspergillus. We elaborate on the physiological importance of tightly regulated endocytosis for cellular fitness under dynamic conditions found in nature and highlight how further understanding and engineering of this process is essential to maximize titer, rate and yield (TRY)-values of engineered cell factories in industrial biotechnological processes.
Molecular and Cellular Biology, 2007
K ؉ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K ؉ transporter, Trk1, is inhibited by high intracellular K ؉ levels and positively regulated by two redundant "halotolerance" protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K ؉ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K ؉ , but not Na ؉ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K ؉ conditions and, in the case of Can1, are stabilized by high K ؉ concentrations. Other plasma membrane proteins such as Pma1 (H ؉ -pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K ؉ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K ؉ and halotolerance kinases on membrane trafficking and/or on the transporters themselves.
Arrestin-Mediated Endocytosis of Yeast Plasma Membrane Transporters
Traffic, 2009
Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.
2019
Nutrient transporters are believed to traffic from their site of synthesis, the ER, to the plasma membrane, through the Golgi, using the conventional vesicular trafficking pathway. However, here we report that the UapA purine transporter in Aspergillus nidulans, follows a Golgi-independent, unconventional new route, which does not involve key Rab GTPases, AP adaptors, microtubules or endosomes, but is dependent on functional COPII, clathrin heavy chain (ClaH) and actin. The role of ClaH in transporter secretion is shown to be unrelated to that performing, together with Rab11/AP-1, at the Golgi, and is seemingly due to an effect in actin network functioning. Our findings are discussed within the frame of a model that rationalizes why the trafficking mechanism uncovered herein might hold true for transporters in higher organisms.One Sentence SummaryFungal transporters use a non-polar, COPII- and actin-dependent, route for subcellular traffic to the cell membrane.
Proceedings of the National Academy of Sciences
The eukaryotic plasma membrane is compartmentalized into domains enriched in specific lipids and proteins. However, our understanding of the molecular bases and biological roles of this partitioning remains incomplete. The best-studied domain in yeast is the membrane compartment containing the arginine permease Can1 (MCC) and later found to cluster additional transporters. MCCs correspond to static, furrow-like invaginations of the plasma membrane and associate with subcortical structures named “eisosomes” that include upstream regulators of the target of rapamycin complex 2 (TORC2) in the sensing of sphingolipids and membrane stress. However, how and why Can1 and other nutrient transporters preferentially segregate in MCCs remains unknown. In this study we report that the clustering of Can1 in MCCs is dictated by its conformation, requires proper sphingolipid biosynthesis, and controls its ubiquitin-dependent endocytosis. In the substrate-free outward-open conformation, Can1 accumu...