The availability of key nutrients, such as amino acids, nitrogen compounds, and sugars, dictate the developmental programs and growth rates of yeast cells. Several overlapping signalling networks including the Ras/cAMP/PKA, AMP-activated kinase, and the TOR1 complex, apprise cells on nutrient availability and influence the cells transcriptional, translational, posttranslational, and metabolic profiles as well as dictating cell fate.
Much of the glucose-induced signalling in yeast functions through the Ras/cAMP/PKA pathway. 90 % of the transcriptional changes that occur on the addition of glucose to glucose depressed cells can be recapitulated by activating this pathway. Conversely, inhibiting this pathway concurrent with glucose addition eliminates most of the glucose induced responses.
54
Thus, Ras and the PKA pathway is necessary for the majority of the transcription changes of the cell in response to glucose addition [170]. Considering the Ras proteins role in glucose signalling it would not be wrong to postulate that Ras may function within the processes underpinning other nutritional sensing pathways in yeast, including peptide transport.
1.14.1 Di/Tri-peptide uptake mechanisms in S. cerevisiae
Peptides are composed of sequences of amino acids and once catabolized provide the essential building blocks required for protein synthesis. In addition, certain peptides and amino acids can be utilized as a nitrogen source and can act as signalling molecules that alter cell behaviour. For example, the branched-chain amino acid leucine, has been shown to control TORC1 activity [171][172]. TORC1 senses and responds to nutrients to promote cell growth and the inhibition of catabolic processes, such as autophagy. Branched-chain amino acids such as leucine function by affecting the ‘nucleotide binding status of the Exit from G 0 Complex’ (EGOC) GTPase subunits Gtr1 and Gtr2 in Saccharomyces cerevisiae
[173][174]. Amongst other TORC1-stimulating amino acids, arginine abundance in mammalian cells is proposed to be sensed by the lysosomal transporter SLC38A9 and conveyed to mTORC1 via the Rag GTPases [175][176], while glutamine levels appear to be transduced to control both yeast and mammalian TORC1 independently of the EGO/Rag GTPases [172].
In mammals, amino acids exert a major role on the regulation of protein synthesis through the control of the kinases mTOR and Gcn2, which have been shown to have opposing effects on protein synthesis [172][177]. The regulation of Gcn2 activity by amino acid availability relies on the capacity of Gcn2 to sense the increased levels of uncharged tRNAs upon amino acid scarcity [177]. Gcn2 is activated during scarcity of an essential amino acids and
55
phosphorylates the α-subunit of eukaryotic initiation factor 2 alpha (eIF2α) [178]. This leads to the general inhibition of protein synthesis.
Due to the multifaceted role of amino acids and peptides in biological systems, it is therefore not surprising that microbes have evolved a multitude of mechanisms to facilitate peptide and amino acid uptake. The study of such mechanisms has been extensively examined in yeast, with amino acid uptake mechanisms presenting high conservation in mammals.
Saccharomyces cerevisiae has two distinct peptide transport mechanisms, a di-/tripeptide (the PTR system) and a tetra-/pentapeptides (the OPT) transport system. The PTR family of peptide transporters transport a number of substrates, including amino acids, nitrates and di/tri-peptides [179]. PTR2 encodes an integral membrane protein (Ptr2p) involved in the physical translocation of peptides across the plasma membrane.
PTR2 encodes an integral membrane protein of 601 amino acids containing 12 membrane- spanning domains which transport substrates by the means of proton-motive force [180]. The transporter is specific for di-/tripeptides, with a preference for peptides containing hydrophobic amino acids. In S. cerevisiae the regulation of PTR2 expression is strongly affected by the composition of the extracellular environment. In the absence of the preferred nitrogen sources PTR2 expression is induced [181][182].The import of di/tripeptides composed of basic or bulky hydrophobic N-terminal residues increases PTR2 expression via reducing cellular levels of Cup9p, the homeodomain-containing transcriptional repressor of PTR2. Specific di/tri-peptides function as both as ligands and regulators of the E3 ubiquitin ligase Ubr1p. Ubr1p mediates Cup9p degradation system that is governed by the identity of N-terminal amino acids [183][184]. Most di/tripeptides are too small to be degraded by the proteasome and are assimilated as nutrients by intracellular peptidases. However,
56
di/tripeptides with basic (Type 1: His, Lys, or Arg) and bulky (Type 2: Ile, Trp, Leu, Tyr, or Phe) N-terminal residues can compete with larger protein substrates and bind at the Type 1 and Type 2 Ubr1p substrate-binding sites. Once bound, Ubr1p-mediated degradation of Cup9p is allosterically activated via the release of the Ubr1p auto inhibitory domain, revealing a substrate-binding domain that binds an internal degron in Cup9p [185]. Relief of Cup9p repression of PTR2 results in enhanced PTR2 expression. A positive regulatory feedback loop is created where di/tripeptide uptake perpetuates Ubr1p-mediated Cup9p degradation, upregulating PTR2 expression and thus increased di/tripeptide uptake.
Although Ptr2p is the major transporter of di/tripeptides in S. cerevisiae, di/tripeptides can also be imported with a low efficacy by Dal5, whose primary function is the import of nitrogen sources, such as allantoate and ureidosuccinate [186]. As aforementioned, the peptide transporters Opt1 and Opt2, which have partially overlapping functions, import peptides of 4–5 residues. In addition, Opt1 is a high affinity importer of glutathione, a “noncanonical” tripeptide [187]. In the same way as the Ptr2 transporter of di/tripeptides, the expression of OTP2 is down-regulated by Cup9p, whereas the expression of OPT1 is independent of Cup9p [187]. In addition to PTR2 and OPT2, the N-end rule pathway also controls the expression of DAL5, but in a manner contrary to that of the other two transporters: whereas Cup9p is a transcriptional repressor of PTR2 and OPT2, Cup9p up-regulates the expression of DAL5 [179][187].
57