ABSTRACTThe peptide transporter Ptr2 plays a central role in di- or tripeptide import inSaccharomyces cerevisiae. AlthoughPTR2transcription 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 ofStreptococcus thermophiluspeptide transporter PepTstand 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 PepTstare involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enableptr2Δtrp1cell 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.
Structural basis of Naa20 activity towards a canonical NatB substrate
