Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.
The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.
In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a ϳ35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH 4 ؉ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake. Ammonium transporters (AMTs)1 of the AMT/MEP/Rh protein family have been identified in all domains of life, including plants, bacteria, archea, yeast, and animals (1, 2). AMT/ MEP/Rh proteins are highly hydrophobic membrane proteins with a predicted molecular mass of ϳ45-55 kDa and 11 or 12 putative transmembrane spans. Initially AMT/MEP/Rh ammonium transporters from yeast and plants were identified molecularly by functional complementation of a yeast mutant defective in ammonium uptake (3-5). Later, homologs were isolated from bacteria (6) and animals (Caenorhabditis elegans), and phylogenetic analysis showed that mammalian Rh (rhesus) blood group polypeptides belong to the same superfamily (7). Heterologously expressed RhAG and a homolog from kidney (RhGK ϭ RhCG) were also shown to function as ammonium transporters (8, 9).Plants require transporters for NH 4 ϩ acquisition from a wide range of external concentrations and are able to concentrate and transiently accumulate NH 4 ϩ in the cytosol before being metabolized or further compartmentalized (10). Ammonium transport across root plasma membranes is biphasic, consisting of a high-affinity and a low-affinity nonsaturating component (11,12). The high-affinity transport system, which operates predominantly at low external ammonium concentrations, is energized by the membrane potential. In tomato, the NH 4 ϩ -uniporter LeAMT1;1 encodes a component of the high-affinity transport system that depends on the membrane potential (13). The molecular identity of the low-affinity transport system, however, is less clear. It contributes significantly to overall NH 4 ϩ uptake at higher external ammonium concentrations (Ͼ1 mM) and may have a distinct transport mechanism, because uncharged NH 3 or charged NH 4 ϩ may be the substrate (11, 12, 14,...
Saccharomyces cerevisiae cells possess a plasma membrane sensor able to detect the presence of extracellular amino acids and then to activate a signaling pathway leading to transcriptional induction of multiple genes, e.g., AGP1, encoding an amino acid permease. This sensing function requires the permease-like Ssy1 and associated Ptr3 and Ssy5 proteins, all essential to activation, by endoproteolytic processing, of the membranebound Stp1 transcription factor. The SCF Grr1 ubiquitin-ligase complex is also essential to AGP1 induction, but its exact role in the amino acid signaling pathway remains unclear. Here we show that Stp1 undergoes casein kinase I-dependent phosphorylation. In the yck mutant lacking this kinase, Stp1 is not cleaved and AGP1 is not induced in response to amino acids. Furthermore, we provide evidence that Ssy5 is the endoprotease responsible for Stp1 processing. Ssy5 is significantly similar to serine proteases, its self-processing is a prerequisite for Stp1 cleavage, and its overexpression causes inducer-independent Stp1 cleavage and high-level AGP1 transcription. We further show that Stp1 processing also requires the SCF Grr1 complex but is insensitive to proteasome inhibition. However, Stp1 processing does not require SCF Grr1 , Ssy1, or Ptr3 when Ssy5 is overproduced. Finally, we describe the properties of a particular ptr3 mutant that suggest that Ptr3 acts with Ssy1 in amino acid detection and signal initiation. We propose that Ssy1 and Ptr3 form the core components of the amino acid sensor. Upon detection of external amino acids, Ssy1-Ptr3 likely allows-in a manner dependent on SCF Grr1 -the Ssy5 endoprotease to gain access to and to cleave Stp1, this requiring prior phosphorylation of Stp1 by casein kinase I.
SummaryRegulation of arginine metabolism requires the integrity of four regulatory proteins, ArgRI, ArgRII, ArgRIII and Mcm1. To characterize further the interactions between the different proteins, we used the twohybrid system, which showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRIII, without an arginine requirement. To de®ne the interacting domains of ArgRI and Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA-binding domain of Gal4 (GBD) and created mutations in GBD-ArgRI and GBD-Mcm1. The putative a helix present in the MADS-box domain of ArgRI and Mcm1 is their major region of interaction with ArgRIII. Interactions between the two MADS-box proteins and ArgRIII were con®rmed using af®nity chromatography. The requirement for ArgRIII in the control of arginine metabolism can be bypassed in vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that ArgRIII is not present in the protein complex formed with the`arginine boxes'. We show that the impairment of arginine regulation in an argRIII deletant strain is a result of a lack of stability of ArgRI and Mcm1. A mutation in ArgRI, impairing its interaction with ArgRIII, leads to an unstable ArgRI protein in a wild-type strain. ArgRIII integrity is crucial for Mcm1 function, as shown by the marked decreased expression of ®ve genes controlled by Mcm1. However, ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression of Mcm1 does not compensate some physiological defects observed in an argRIII deletant strain.
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