Peter Poulsen

Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p

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 acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand.

Mapping of an internal protease cleavage site in the Ssy5p component of the amino acid sensor of Saccharomyces cerevisiae and functional characterization of the resulting pro- and protease domains by gain-of-function genetics.

ABSTRACT Ssy5p is a 77-kDa protein believed to be a component of the SPS amino acid sensor complex in the plasma membrane of Saccharomyces cerevisiae. Ssy5p has been suggested to be a chymotrypsin-like serine protease that activates the transcription factor Stp1p upon exposure of the yeast to extracellular amino acid. Here we overexpressed and partially purified Ssy5p to improve our understanding of its structure and function. Antibodies against Ssy5p expressed in Escherichia coli were isolated and used to detect Ssy5p processing in S. cerevisiae cells. Partial purification and N-terminal sequencing of processed Ssy5p revealed in vivo cleavage of Ssy5p between amino acids 381 and 382. We also isolated constitutively signaling SSY5 mutants and quantified target promoter activation and Stp1p processing. One mutant contained an amino acid substitution in the prodomain, whereas three others harbored amino acid substitutions in the protease domain. Dose-response analysis indicated that all four mutants exhibited increased basal levels of Stp1p processing. Interestingly, whereas the three constitutive mutants mapping to the protease domain of Ssy5p exhibited the decreased 50% effective concentration (EC50) characteristic of constitutive mutations previously found in Ssy1p, Ptr3p, and Ssy5p, the EC50 of the mutation that maps to the prodomain of Ssy5p remained essentially unchanged. In a model of Ssy5p derived from its similarities with α-lytic protease from Lysobacter enzymogenes, the sites corresponding to the mutations in the protease domain are clustered in a region facing the prodomain, suggesting that this region interacts with the prodomain and participates in the conformational dynamics of sensing.

Effect of UTP and GTP pools on attenuation at the pyrE gene of Escherichia coli

SummaryWe have used the galK gene, minus its promoter, to quantitate transcription of the orfE-pyrE operon of Escherichia coli in front of and after the intercistronic attenuator. Expression of the hybrid genes was studied in a bacterium with mutations that permit changes in the UTP and GTP pools during exponential growth. It was found that the greater part of pyrE gene regulation by the nucleotides takes place at the intercistronic attenuator and that promoter control contributes only little, ca. twofold. When pools of both UTP and GTP were high only 5%–6% of the mRNA chains were continued into the pyrE gene. However, when the UTP pool was reduced (from 1.3 to 0.2 μmol/g dry weight) nearly 100% of transcription passed the attenuator. Likewise, a reduction in the GTP pool (from 3.2 to 0.8 μmol/g dry weight) resulted in 25%–30% escape of attenuation. Regulation by attenuation disappeared when a premature stop-codon was introduced near the end of orfE such that translational coupling to transcription was prevented in the attenuator area. Therefore, we attribute the modulation of attenuation to nucleotide-induced variations in the kinetics of mRNA chain elongation. In support for this it was found that an RNA polymerase mutant with reduced RNA chain growth rate transcribed past the pyrE attenuator at a high frequency in the presence of a high UTP pool, but only when coupling of translation to transcription was allowed at the end of orfE.

Hyper- and hyporesponsive mutant forms of the Saccharomyces cerevisiae Ssy1 amino acid sensor

The Saccharomyces cerevisiae integral membrane protein Ssy1p functions with Ssy5p and Ptr3p to sense extracellular amino acids. Signal transduction leads to processing and nuclear localization of Stp1p and Stp2p, transcriptional activators of many amino acid transporter genes. Ssy1p is structurally related to amino acid permeases, but unable to transport amino acids. We isolated SSY1 mutants that constitutively activate a target promoter. Dose-response analysis showed that the mutants are hyperresponsive, requiring less inducer to give strong signaling than does the wild type. Another mutant (Ssy1pT639I) turned out to be hyporesponsive, i.e., it signals only at high inducer concentration. In accordance with a transporter-like mechanism for Ssy1p function we suggest that the hyper- and hyporesponsive mutant forms differ from the wild-type sensor by being more and less inclined, respectively, to adopt an outward-facing, signaling conformation. Coordinate conformational dynamics of the sensor complex was supported by additive effects of combinations of constitutive SSY1, PTR3 and SSY5 alleles. Assuming structural similarity of Ssy1p to the distantly related bacterial leucine transporter LeuTAa, several activating substitutions were located near the substrate binding site while others were on the periphery of Ssy1p. We suggest analyses of transporter-like sensors as an approach to understand key features of transporters.

Attenuation at nucleotide biosynthetic genes and amino acid biosynthetic operons of Escherichia coli

Abstract Transcription and translation are physically coupled in bacteria. The coupling is variable and regulates expression of many genes. It influences premature termination of mRNA chain synthesis— a process termed attenuation. In some cases attenuation is controlled primarily by the saturation of RNA polymerase with nucleotides, at other genes the concentration of aminoacyl-tRNAs is of primary importance.

Role of transcription pausing in the control of the pyrE attenuator in Escherichia coli.

Expression of the Escherichia coli pyrE gene is regulated by transcription attenuation in the intercistronic orfE–pyrE region and modulated by the distance between the transcribing RNA polymerase and the leading ribosome as a function of the supply of UTP and GTP. In this communication we show that pyrE expression is hyper‐repressed in vivo following addition of uracil in strains carrying the nusAcs10 mutation. This phenotype, previously seen in rpsL 1204 strains whose ribosomes are pseudodependent on streptomycin and work at suboptimal elongation rate, indicates that RNA polymerase escapes from the ribosomes in the pyrE attenuator region in the nusA mutant. In vitro transcription studies revealed that the build‐up of the full‐length attenuated orfE transcript occurred more slowly in the presence of the NusA protein than in its absence. Moreover, the NusA protein enhanced several transcription pauses through the orfE gene. These effects were more pronounced when low concentrations of either UTP or GTP were used than at low concentrations of either CTP or ATP. The results indicate that the NusA protein is required for proper regulation of pyrE gene expression and is involved, together with the NTP pools, in maintaining the coupling between transcription and translation in the pyrE attenuator region by inhibiting RNA chain elongation.

Constitutive Signal Transduction by Mutant Ssy5p and Ptr3p Components of the SPS Amino Acid Sensor System in Saccharomyces cerevisiae

ABSTRACT Amino acids in the environment of Saccharomyces cerevisiae can transcriptionally activate a third of the amino acid permease genes through a signal that originates from the interaction between the extracellular amino acids and an integral plasma membrane protein, Ssy1p. Two plasma membrane-associated proteins, Ptr3p and Ssy5p, participate in the sensing, which results in cleavage of the transcription factors Stp1p and Stp2p, removing 10 kDa of the N terminus of each of them. This confers the transcription factors with the ability to gain access to the nucleus and activate transcription of amino acid permease genes. To extend our understanding of the role of Ptr3p and Ssy5p in this amino acid sensing process, we have isolated constitutive gain-of-function mutants in these two components by using a genetic screening in which potassium uptake is made dependent on amino acid signaling. Mutants which exhibit inducer-independent processing of Stp1p and activation of the amino acid permease gene AGP1 were obtained. For each component of the SPS complex, constitutive signaling by a mutant allele depended on the presence of wild-type alleles of the other two components. Despite the signaling in the absence of inducer, the processing of Stp1p was more complete in the presence of inducer. Dose response assays showed that the median effective concentration for Stp1p processing in the mutant cells was decreased; i.e., a lower inducer concentration is needed for signaling in the mutant cells. These results suggest that the three sensor components interact intimately in a complex rather than in separate reactions and support the notion that the three components function as a complex.

Molecular and mutational analysis of three genes preceding pyrE on the Escherichia coli chromosome

The nucleotide sequence of two kilobase pairs (kb) 5′ to the orfE‐pyrE operon has been determined. The sequence revealed two open reading frames, orfX and orfY, consisting of 286 and 274 codons, respectively, and having a transcriptional orientation opposite that of the orfE‐pyrE operon. Analysis of transcription initiations showed that the promoters of orfE and orfX constitute a pair of divergent promoters with overlapping ‐35 regions and that orfY is transcribed from an independent promoter. Translational analysis indicated that the orfs are expressed in Escherichia coli.

Crystallization and preliminary X-ray analysis of maltose O-acetyltransferase

Maltose O-acetyltransferase (Mac) is a member of the hexapeptide-repeat family of enzymes, which contains proteins with left-handed parallel beta-helix architecture forming homotrimers. Diffraction data for four well diffracting crystal forms were collected. Crystal form I diffracted beyond 1.53 A resolution but was perfectly merohedrally twinned with an apparent space group P622. Crystal forms II and III (space groups R3 and C2, respectively) could be obtained under very similar conditions by adjusting the buffer pH differently. Crystal forms II and III had several monomers in the asymmetric unit and were difficult to derivatize. However, during soaking with trimethyl lead acetate, the form III crystals dissolved and crystals with a different habit and space group grew in their place (form IV). In three of the crystal forms, a ladder of peaks was visible in the native Patterson maps along the c axis. These peaks were interpreted as corresponding to the vectors between the beta-strands in the turns of the beta-helix. Crystal form IV is suitable for structure determination of Mac exploiting the anomalous scattering of lead.