Membrane proteins associated with amino acid transport by yeast (Saccharomyces cerevisiae).

Cells of the wild-type yeast (Saccharomyces cerevisiae) strain Y185, grown under conditions that de-repress the formation of a general amino acid permease ('Gap') system, bind delta-N-chloroacetyl[1-(14)C]ornithine; L- and D-amino acid substrates of the general amino acid permease system protect against this binding. The protein responsible is released from the cells by homogenization or by preparation of protoplasts; it is not released by osmotic shock. This protein is virtually absent from the wild-type strain when it is grown under conditions that repress the general amino acid permease system, and is also absent from a Gap- mutant Y185-His3, selected by its resistance to D-amino acids. This mutant and repressed wild-type cells also fail to form a number of membrane proteins elaborated by de-repressed wild-type cells. It is possible that all these proteins are components of the general amino acid permease system.     

A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.     

Anilinonaphthalene sulfonate fluorescence and amino acid transport in yeast

Summary Fluorescence of 1-anilinonaphthalene-8-sulfonate in yeast membranes appears to be caused predominantly by binding to lipids (ANS_proteinANS_lipid120) as indicated by the fluorescence lifetime, degree of polarization, and excitation spectra. It was insensitive to short-circuiting the membrane potential. Fluorescence intensity increased as cells (especially after pretreatment with energy donors such as glucose) were exposed to some amino acids, in particular, aspartic and glutamic acids. The character of fluorescence shifted to that of protein-bound ANS, suggesting an exposure of new protein sites accessible to the probe. This shift could be prevented by inhibitors of energy transduction as well as of transport. TheK _1/2 of the shift was at 2.5mm aspartic acid.     

Effect of phosphatidylserine enrichment on amino acid transport in yeast

A 1.5- to 3.5-fold accumulation of phosphatidylserine was observed when Candida albicans and Saccharomyces cerevisiae cells were grown in the presence of hydroxylamine, a known inhibitor of phosphatidylserine decarboxylase. However, as compared to S. cerevisiae cells, the levels of phosphatidylcholine and phosphatidylethanolamine were much lower in C. albicans cells. The enrichment of phosphatidylserine selectively affected the transport of several amino acids.     

Novel families of vacuolar amino acid transporters

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed.     

Mutated-gamma-actin restores growth to a yeast amino acid transport defective mutant

A mutated yeast cell 22574d lacking all three proline transporters, PUT4, UGA4, and GAP1, and incapable of growth on proline recovers its lost ability to grow on proline as sole nitrogen source when transformed with a mutagenized mouse gamma-actin cDNA (M-gamma-A). Native mouse gamma-actin cDNA is ineffective. The 3'-region of gamma-actin cDNA was mutagenized to resemble E51 cDNA previously isolated from Ehrlich tumor cells. The E51 cDNA has an extended reading frame in the 3'-region compared to that in native gamma-actin. The extension of the open reading frame in E51 cDNA, was found to be due to an additional pair of bases (TG) at position 1104 of E51 cDNA. After site-directed mutagenesis of the 3'-region of native gamma-actin cDNA to resemble that of E51 cDNA, the construct, M-gamma-A cDNA, was expressed in the 22574d yeast. While the transformation with M-gamma-A increased the uptake of both proline and gamma-amino butyric acid, the transport of five other solutes was not changed by this transformation. Northern blotting of the nontransformed and the M-gamma-A-transformed 22574d cells with gene-specific probes for the three proline transporters showed the expression of an mRNA for UGA4 in both transformed and nontransformed cells but no evidence for the expression of GAP1 or PUT4. The mRNA for UGA4 was expressed at a lower level in strain 22574d than in the parent yeast sigma1278b. Furthermore, the message in the mutated cells is smaller in size by about 15%. These results are consistent with the synthesis of a mutated transporter which requires the coexpression of M-gamma-A, but not native gamma-actin, to restore physiological function, i.e., proline or gamma-amino acid transport.     

Amino acids regulate retrieval of the yeast general amino acid permease from the vacuolar targeting pathway

Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.     

Specificity of trans-inhibition of amino acid transport in Baker’s yeast

The trans-inhibition potency of intracellular amino acids on the transport of various amino acids follows the same sequence,viz. Pro(Lys), Phe, Glu, Ala, Gly, Leu, and α-aminoisobutyric acid. The same sequence was found for the reciprocal of trans-inhibition constants. It appears that the intracellular amino acid itself or a derivative thereof acts on a component that is common to all amino acid transport systems of baker’s yeast.     

Chloride transport of yeast vacuolar membrane vesicles: a study of in vitro vacuolar acidification.

Effects of various solutes on acidification inside the vacuolar membrane vesicles of the yeast Saccharomyces cerevisiae were examined. ATP-dependent acidification was stimulated by the presence of chloride salts. There was essentially no difference in the stimulatory effects of NaCl, KCl, LiCl, and choline chloride. The membrane potential across the vacuolar membrane was reduced by the presence of Cl- salts. Transport of 36Cl- is driven by the protonmotive force across the vacuolar membrane. Kinetic analyses have revealed that the stimulatory effect of Cl- on internal acidification depends on two distinct components. One shows linear dependency on chloride concentration and is inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid (DIDS). The other exhibits saturable kinetics with an apparent Km for chloride of 15-20 mM. We conclude that the vacuolar membrane of yeast is equipped with Cl- transport systems contributing to the formation of a chemical gradient of protons across the vacuolar membrane by shunting the membrane potential generated by proton translocation.     

A new family of yeast nuclear pore complex proteins

We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.     

A single amino acid change in the yeast vacuolar metal transporters ZRC1 and COT1 alters their substrate specificity

Iron is an essential nutrient but in excess may damage cells by generating reactive oxygen species due to Fenton reaction or by substituting for other transition metals in essential proteins. The budding yeast Saccharomyces cerevisiae detoxifies cytosolic iron by storage in the vacuole. Deletion of CCC1, which encodes the vacuolar iron importer, results in high iron sensitivity due to increased cytosolic iron. We selected mutants that permitted Deltaccc1 cells to grow under high iron conditions by UV mutagenesis. We identified a mutation (N44I) in the vacuolar zinc transporter ZRC1 that changed the substrate specificity of the transporter from zinc to iron. COT1, a vacuolar zinc and cobalt transporter, is a homologue of ZRC1 and both are members of the cation diffusion facilitator family. Mutation of the homologous amino acid (N45I) in COT1 results in an increased ability to transport iron and decreased ability to transport cobalt. These mutations are within the second hydrophobic domain of the transporters and show the essential nature of this domain in the specificity of metal transport.     

A yeast with unusual sulphur amino acid metabolism

A yeast strain highly resistant to propargylglycine (an inhibitor of cystathionine gamma-lyase) was isolated from air. It was partially characterized, but it has not been identified with any known yeast species. Its sulphur amino acid metabolism differed from that of other fungi by the lack of the reverse transsulphuration pathway from methionine to cysteine, as no activity of cystathionine beta-synthase or cystathionine gamma-lyase was found. The functional lack of this pathway was confirmed by growth tests and by experiments with [35S]methionine. In contrast to Saccharomyces cerevisiae neither homocysteine synthase nor the sulphate assimilation pathway were repressible by methionine in the new strain; on the contrary, a regulatory effect of cysteine was observed.     

Carbohydrate-binding proteins in bovine kidney have consensus amino acid sequences of annexin family proteins.

Ca(2+)-dependent carbohydrate-binding proteins were purified from bovine kidney extracts. Upon SDS-polyacrylamide gel electrophoresis under nonreducing conditions, the purified fraction gave doublet protein bands corresponding to 33 kDa (p33) and 41 kDa (p41). Under reducing conditions, a single protein band (p33) was observed. p33 and p41 were submitted to proteolytic digestion with endoproteinase Lys-C, the peptides produced were separated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined by an automated gas-phase protein sequenator. Most of the resulting partial amino acid sequences of these proteins were strikingly homologous to annexin IV, an annexin family protein, i.e. Ca2+/phospholipid-binding proteins, especially in the consensus sequences. In the presence of Ca2+, both proteins bound to vesicles composed of phosphatidylserine and phosphatidylethanolamine, but not phosphatidylcholine. These results indicated that p33 and p41 are members of annexin family proteins.     

Vba2p, a vacuolar membrane protein involved in basic amino acid transport in Schizosaccharomyces pombe

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.     

A novel system A isoform mediating Na+/neutral amino acid cotransport

A cDNA clone encoding a plasma membrane alanine-preferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen. No signal is detected in liver or testis. In the central nervous system, SAT2 is expressed in neurons. SAT2 is significantly up-regulated during differentiation of cerebellar granule cells and is absent from astrocytes in primary culture. The functional properties of SAT2, examined using transfected fibroblasts and in cRNA-injected voltage-clamped Xenopus oocytes, show that small aliphatic neutral amino acids are preferred substrates and that transport is voltage- and Na(+)-dependent (1:1 stoichiometry), pH-sensitive, and inhibited by alpha-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of system A. Kinetic analyses of alanine and MeAIB uptake by SAT2 are saturable, with Michaelis constants (K(m)) of 200-500 microm. In addition to its ubiquitous role as a substrate for oxidative metabolism and a major vehicle of nitrogen transport, SAT2 may provide alanine to function as the amino group donor to alpha-ketoglutarate to provide an alternative source for neurotransmitter synthesis in glutamatergic neurons.     

A new family of integral membrane proteins involved in transport of aromatic amino acids in Escherichia coli.

The nucleotide sequence of tnaB of the tryptophanase operon of Escherichia coli is presented. TnaB is a tryptophan-specific permease that is homologous to Mtr, a second tryptophan-specific permease, and to TyrP, a tyrosine-specific permease. Each member of this family appears to contain 11 membrane-spanning domains.