Regulation of Amino Acid Transport in Saccharomyces cerevisiae

SUMMARYWe review the mechanisms responsible for amino acid homeostasis in Saccharomyces cerevisiae and other fungi. Amino acid homeostasis is essential for cell growth and survival. Hence, the de novo synthesis reactions, metabolic conversions, and transport of amino acids are tightly regulated. Regulation varies from nitrogen pool sensing to control by individual amino acids and takes place at the gene (transcription), protein (posttranslational modification and allostery), and vesicle (trafficking and endocytosis) levels. The pools of amino acids are controlled via import, export, and compartmentalization. In yeast, the majority of the amino acid transporters belong to the APC (amino acid-polyamine-organocation) superfamily, and the proteins couple the uphill transport of amino acids to the electrochemical proton gradient. Although high-resolution structures of yeast amino acid transporters are not available, homology models have been successfully exploited to determine and engineer the catalytic and regulatory functions of the proteins. This has led to a further understanding of the underlying mechanisms of amino acid sensing and subsequent downregulation of transport. Advances in optical microscopy have revealed a new level of regulation of yeast amino acid transporters, which involves membrane domain partitioning. The significance and the interrelationships of the latest discoveries on amino acid homeostasis are put in context

Roles of members of the conserved DedA/Tvp38 membrane protein family in Escherichia coli drug resistance and alkaline pH tolerance

The objective of this dissertation is to understand the functions of Escherichia coli YqjA and YghB, which are members of the conserved DedA/Tvp38 membrane protein family. YqjA and YghB are inner membrane (IM) proteins with multiple predicted membrane–spanning domain, sharing 62% amino acid identity and partly overlapping functions. Simultaneous in-frame deletions of these two genes in a strain named BC202 results in various phenotypes including cell division defects, temperature sensitivity, and sensitivity to drugs and alkaline pH. The cell division defect of BC202 is due to the inefficient secretion of periplasmic amidases by the twin arginine transport (Tat) pathway into the periplasm and drug sensitivity is due to the inefficient function of various drug efflux pumps. Both these phenotypes are related to the loss of proton motive force (PMF) in BC202. Overexpression of MdfA, a Na+-K+/H+ antiporter or growth in acidic media has the ability to rescue all the phenotypes of BC202. In addition, the ∆yqjA mutant (but not the ∆yghB mutant) is alkaline sensitive and overexpression of yqjA can restore growth at alkaline pH only when more than 100mM of sodium or potassium ions is present in the growth medium. Osmotic pressure augments the YqjA mediated growth at alkaline pH. Furthermore, charged amino acids are also essential for YqjA and YghB function that were previously shown important for various secondary transporters. Additionally, yqjA expression is higher at alkaline pH and increased expression of yqjA also required sodium/potassium salts above pH 9.0. The transcriptional regulator CpxR is required for the expression of yqjA at alkaline pH in the presence of Na+/K+. Based on these results, we suggest YqjA and YghB are osmosensing proton-dependent transporters required for E. coli drug resistance and alkaline pH tolerance

Molecular determinants for the thermodynamic and functional divergence of uniporter GLUT1 and proton symporter XylE

<div><p>GLUT1 facilitates the down-gradient translocation of D-glucose across cell membrane in mammals. XylE, an <i>Escherichia coli</i> homolog of GLUT1, utilizes proton gradient as an energy source to drive uphill D-xylose transport. Previous studies of XylE and GLUT1 suggest that the variation between an acidic residue (Asp27 in XylE) and a neutral one (Asn29 in GLUT1) is a key element for their mechanistic divergence. In this work, we combined computational and biochemical approaches to investigate the mechanism of proton coupling by XylE and the functional divergence between GLUT1 and XylE. Using molecular dynamics simulations, we evaluated the free energy profiles of the transition between inward- and outward-facing conformations for the <i>apo</i> proteins. Our results revealed the correlation between the protonation state and conformational preference in XylE, which is supported by the crystal structures. In addition, our simulations suggested a thermodynamic difference between XylE and GLUT1 that cannot be explained by the single residue variation at the protonation site. To understand the molecular basis, we applied Bayesian network models to analyze the alteration in the architecture of the hydrogen bond networks during conformational transition. The models and subsequent experimental validation suggest that multiple residue substitutions are required to produce the thermodynamic and functional distinction between XylE and GLUT1. Despite the lack of simulation studies with substrates, these computational and biochemical characterizations provide unprecedented insight into the mechanistic difference between proton symporters and uniporters.</p></div