Functional Implications and Ubiquitin-Dependent Degradation of the Peptide Transporter Ptr2 in Saccharomyces cerevisiae

Abstract

The peptide transporter Ptr2 plays a central role in di-or tripeptide import in Saccharomyces cerevisiae. Although PTR2 transcription has been extensively analyzed in terms of upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this transporter is limited. Here we identified 14 amino acid residues required for peptide import through Ptr2 based on the crystallographic information of Streptococcus thermophilus peptide transporter PepT st and based on the conservation of primary sequences among the proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 mutations of which the corresponding residues of PepT st are involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enable ptr2⌬trp1 cell growth in alanyl-tryptophan (Ala-Trp) medium. We observed that Ptr2 underwent rapid degradation after cycloheximide treatment (half-life, approximately 1 h), and this degradation depended on Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, W362L, and E480D) exhibited severe defects in peptide import and were subjected to Rsp5-dependent degradation when cells were moved to Ala-Trp medium, whereas, similar to what occurs in the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results suggest that Ptr2 undergoes quality control and the defects in peptide binding and the concomitant conformational change render Ptr2 subject to efficient ubiquitination and subsequent degradation.

Study of the Plasma Membrane Proteome Dynamics Reveals Novel Targets of the Nitrogen Regulation in Yeast

“…Such a combination has been identified for the stress-induced endocytosis of Gap1 that can be mediated by Bul1, Bul2, Aly1/Art6, or Aly2/Art3 (43). Recently, Kawai and colleagues (44) documented that the addition of cycloheximide to cells grown in synthetic dextrose (S.D.) medium triggers Rsp5-dependent ubiquitylation and endocytosis of Ptr2.…”

New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations

“…This finding helped to explain why cycloheximide had little effect on amino acid secretion (Figure B), even though one would expect significant depletion of Ptr2p after 90 min with no protein synthesis [the natural lifetime of Ptr2p is about 1 h (Kawai et al ., )]. Apparently the peptides responsible for all secreted leucine were loaded in the cell in the first 30 min, and it was the amount of active Ptr2p in this time window that determined how much secreted leucine was observed at the end of the 90 min interval.…”

“…Less is known about post-transcriptional regulation of PTR2, although several genes involved in maturation of PTR2 mRNA and in Ptr2p trafficking have been identified (Cai et al, 2006). It was also shown that, when Ptr2p expression is induced with amino acids, the protein undergoes rapid turnover mediated by ubiquitylation and degradation (Kawai et al, 2014).…”

ESCRT-Independent Surface Receptor And Transporter Protein Degradation By The ILF Pathway

“…Upon reexamination of micrographs presented in earlier reports on receptor and transporter down-regulation, we found that some internalized surface polytopic proteins appear on lysosome membranes en route to the lumen for degradation, e.g. the high affinity tryptophan permease Tat2 ( Beck et al, 1999 ), glucose transporters Hxt1 and Hxt3 ( O’Donnell et al, 2015 ), peptide transporter Ptr2 ( Kawai et al, 2014 ), and myo-inositol transporter Itr1 ( Nikko and Pelham, 2009 ). We also noticed that most of these published studies do not directly assess whether ESCRTs are required for protein degradation.…”