Ammonium is a nitrogen source supporting growth of yeast cells at an optimal rate. We recently reported the first characterization of an NH 4 ؉ transport protein (Mep1p) in Saccharomyces cerevisiae. Here we describe the characterization of two additional NH 4 ؉ transporters, Mep2p and Mep3p, both of which are highly similar to Mep1p. The Mep2 protein displays the highest affinity for NH 4 ؉ (K m , 1 to 2 M), followed closely by Mep1p (K m , 5 to 10 M) and finally by Mep3p, whose affinity is much lower (K m , ϳ1.4 to 2.1 mM). A strain lacking all three MEP genes cannot grow on media containing less than 5 mM NH 4 ؉ as the sole nitrogen source, while the presence of individual NH 4 ؉ transporters enables growth on these media. Yet, the three Mep proteins are not essential for growth on NH 4 ؉ at high concentrations (>20 mM). Feeding experiments further indicate that the Mep transporters are also required to retain NH 4 ؉ inside cells during growth on at least some nitrogen sources other than NH 4 ؉ . The MEP genes are subject to nitrogen control. In the presence of a good nitrogen source, all three MEP genes are repressed. On a poor nitrogen source, MEP2 expression is much higher than MEP1 and MEP3 expression. High-level MEP2 transcription requires at least one of the two GATA family factors Gln3p and Nil1p, which are involved in transcriptional activation of many other nitrogen-regulated genes. In contrast, expression of either MEP1 or MEP3 requires only Gln3p and is unexpectedly downregulated in a Nil1p-dependent manner. Analysis of databases suggests that families of NH 4 ؉ transporters exist in other organisms as well.

We have identified the gene DYNI, which encodes the heavy chain of cytoplasmic dynein in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence (Mr 471,305) reveals the presence of four P-loop motifs, as in all dyneins known so far, and has 28% overall identity to the dynein heavy chain ofDictyostelium [Koonce, M. P., Grissom, P. M. & McIntosh, J. R. (1992) J. CeUl Biol. 119, 1597Biol. 119, -1604 with 40% identity in the putative motor domain. Disruption of DYNI causes misalignment of the spindle relative to the bud neck during cell division and results in abnormal distribution of the dividing nuclei between the mother cell and the bud. Cytoplasmic dynein, by generating force along cytoplasmic microtubules, may play an important role in the proper alignment of the mitotic spindle in yeast.Nuclear and cytoplasmic microtubules in the yeast Saccharomyces cerevisiae participate in several well-defined cellular processes that include the segregation of chromosomes and the migration of the nucleus during mitosis as well as the migration and fusion of nuclei in karyogamy (1). These processes are believed to involve microtubule-based motor enzymes, and recently several genes containing sequence regions with homology to the motor domain characteristic of the kinesin superfamily have been identified in S. cerevisiae and other fungi (2-8).The cytoplasmic form ofthe microtubule-associated motor dynein has ATPase activity and moves toward the minus ends of microtubules in in vitro gliding assays (9). Cytoplasmic dynein exists in a wide variety of eukaryotic cells, including Dictyostelium (10, 11), and indirect evidence has suggested that it plays a major role in the movement of chromosomes toward the minus ends of spindle microtubules at the anaphase stage of mitosis (12, 13), as well as in the retrograde transport of organelles in nerve axons (14)(15)(16) Transformations were done with the lithium acetate method (18). Synchronization of yeast cultures was achieved by treating with a mating factor as described by Berlin et al (19).PCR Primers. Oligonucleotide primers were designed from the deduced amino acid sequence of the sea urchin dynein ,B heavy chain (20, 21) in regions that were conserved between sea urchin dynein isoforms (22). The nucleotide sequences of the two degenerate primers that were used to PCR-amplify pDLP1 from yeast genomic DNA are 5'-CCTGCTGGNAC-NGGNAARAC-3' (sense strand, targeting amino acidsequence PAGTGKT) and 5'-TACCCIGGRTTCATIGTDA-TRAA-3' (antisense strand, targeting amino acid-sequence FITMNPG).DNA Sequencing. Nucleotide sequencing of the original probe, pDLP1, and other restriction fragments in the vicinity of the putative hydrolytic ATP-binding site was done by subcloning into M13mpl8/mp19 vectors and using universal M13 primers and a Sequenase 2.0 DNA sequencing kit (United States Biochemical).The nucleotide sequence of the complete gene was determined as part of the ongoing project to sequence the entire chromosome XI in yeast (strain S288C). Relevant cosmids were soni...

The SSY1 gene of Saccharomyces cerevisiae encodes a member of a large family of amino acid permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of the AGP1 gene encoding a low-affinity, broad-specificity amino acid permease. Total noninduction of the AGP1 gene in the ssy1⌬ mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1 requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys 6 -Zn 2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1 by amino acids also requires Grr1p, the F-box protein of the SCF Grr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.

In Saccharomyces cerevisiae, the transport of ammonium across the plasma membrane for use as a nitrogen source is mediated by at least two functionally distinct transport systems whose respective encoding genes are called MEP1 and MEP2. Mutations in the MEP2 gene affect high affinity, low capacity ammonium transport while mutations in the MEP1 gene disrupt a lower affinity, higher capacity system. In this work, the MEP1 gene has been cloned and sequenced and its expression analyzed. The predicted amino acid sequence reveals a highly hydrophobic, 54 kDa protein with 10 or 11 putative membrane‐spanning regions. The predicted Mep1p protein shares high sequence similarity with several bacterial proteins of unknown function, notably the product of the nitrogen‐regulated nrgA gene of Bacillus subtilis, and with that of a partial cDNA sequence derived from Caenorhabditis elegans. The Mep1p and related proteins appear to define a new family of transmembrane proteins evolutionarily conserved in at least bacteria, fungi and animals. The MEP1 gene is most highly expressed when the cells are grown on low concentrations of ammonium or on ‘poor’ nitrogen sources like urea or proline. It is down‐regulated, on the other hand, when the concentration of ammonium is high or when other ‘good’ nitrogen sources like glutamine or asparagine are supplied in the culture medium. The overall properties of Mep1p indicate that it is a transporter of ammonium. Its main function appears to be to enable cells grown under nitrogen‐limiting conditions to incorporate ammonium present at relatively low concentrations in the growth medium.

We compared the transcriptomes of Saccharomyces cerevisiae cells growing under steady-state conditions on 21 unique sources of nitrogen. We found 506 genes differentially regulated by nitrogen and estimated the activation degrees of all identified nitrogen-responding transcriptional controls according to the nitrogen source. One main group of nitrogenous compounds supports fast growth and a highly active nitrogen catabolite repression (NCR) control. Catabolism of these compounds typically yields carbon derivatives directly assimilable by a cell's metabolism. Another group of nitrogen compounds supports slower growth, is associated with excretion by cells of nonmetabolizable carbon compounds such as fusel oils, and is characterized by activation of the general control of amino acid biosynthesis (GAAC). Furthermore, NCR and GAAC appear interlinked, since expression of the GCN4 gene encoding the transcription factor that mediates GAAC is subject to NCR. We also observed that several transcriptional-regulation systems are active under a wider range of nitrogen supply conditions than anticipated. Other transcriptional-regulation systems acting on genes not involved in nitrogen metabolism, e.g., the pleiotropic-drug resistance and the unfolded-protein response systems, also respond to nitrogen. We have completed the lists of target genes of several nitrogen-sensitive regulons and have used sequence comparison tools to propose functions for about 20 orphan genes. Similar studies conducted for other nutrients should provide a more complete view of alternative metabolic pathways in yeast and contribute to the attribution of functions to many other orphan genes.