Genetic and biochemical analysis of the yeast plasma membrane Ssy1p-Ptr3p-Ssy5p sensor of extracellular amino acids

Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment. In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases. We have found that SSY5 encodes a third component of this amino acid sensing system. Mutations in SSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 and ptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations. Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein. Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components. These results suggest that these components function as part of a sensor complex localized to the plasma membrane. Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner. Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids. Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition. The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.     

Ssy1 functions at the plasma membrane as a receptor of extracellular amino acids independent of plasma membrane‐endoplasmic reticulum junctions

Evidence from multiple laboratories has implicated Ssy1, a nontransporting amino acid permease, as the receptor component of the yeast plasma membrane (PM)‐localized SPS (Ssy1‐Ptr3‐Ssy5)‐sensor. Upon binding external amino acids, Ssy1 is thought to initiate signaling events leading to the induction of amino acid permease gene expression. In striking contrast, Kralt et al (2015) (Traffic 16 :135‐147) have questioned the role of Ssy1 in amino acid sensing and reported that Ssy1 is a component of the endoplasmic reticulum (ER), where it reportedly participates in the formation of ER‐PM junctions. Here, we have re‐examined the intracellular location of Ssy1 and tested the role of ER‐PM junctions in SPS sensor signaling. We show that the C‐terminal of Ssy1 carries a functional ER‐export motif required for proper localization of Ssy1 to the PM. Furthermore, ER‐PM junctions are dispensable for PM‐localization and function of Ssy1; Ssy1 localizes to the PM in a Δtether strain lacking ER‐PM junctions (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ), and this strain retains the ability to initiate signals induced by extracellular amino acids. The data demonstrate that Ssy1 functions as the primary amino acid receptor and that it carries out this function at the PM.     

The fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase

Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)+ RNA in the nuclei at the nonpermissive temperature. We found that the ptr1+ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase. In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated. The ptr1+ gene is essential for growth and its mutation did not affect nuclear protein export. A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p. We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2+ gene isolated as a multi-copy suppressor of cdc7-PD1.     

Amino acids control ammonia pulses in yeast colonies

Individual yeast colonies produce pulses of volatile ammonia separated by phases of medium acidification. Colonies of Saccharomyces cerevisiae mutant defective in the general amino acid permease, Gap1p, exhibit decreased ammonia production. Mutations in the S. cerevisiae amino acid sensor SPS completely abolish the colony ammonia pulses. In contrast, the ammonia pulse production is independent of external concentrations of ammonium and of its uptake by the ammonium permeases Mep1p, Mep2p, and Mep3p. It is concluded that in S. cerevisiae colonies, the extracellular amino acids, but not the extracellular ammonium, serve as a source for volatile ammonia production. These phenomena are not restricted to S. cerevisiae, since we observe that extracellular levels of 8 out of the 20 tested amino acids are necessary for ammonia pulses produced by Candida mogii colonies.     

Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae

The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport mechanisms, one for di-/tripeptides (the PTR system) and another for tetra-/pentapeptides (the OPT system). The PTR system consists of three genes, PTR1, PTR2 and PTR3. The transporter (Ptr2p), encoded by the gene PTR2, is a 12 transmembrane domain (TMD) integral membrane protein that translocates di-/tripeptides. Homologues to Ptr2p have been identified in virtually all organisms examined to date and comprise the PTR family of transport proteins. In S. cerevisiae, the expression of PTR2 is highly regulated at the cellular level by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1. Oligopeptides, consisting of four to five amino acids, are transported by the 12-14 TMD integral membrane protein Opt1p. Unlike Ptr2p, distribution of this protein appears limited to fungi and plants, and there appears to be three paralogues in S. cerevisiae. This transporter has an affinity for enkephalin, an endogenous mammalian pentapeptide, as well as for glutathione. Although it is known that OPT1 is normally expressed only during sporulation, to date little is known about the genes and proteins involved in the regulation of OPT1 expression.