Substrate preference is altered by mutations in the fifth transmembrane domain of Ptr2p, the di/tri-peptide transporter of Saccharomyces cerevisiae.
The integral membrane protein Ptr2p transports di/tri-peptides into the yeast Saccharomyces cerevisiae. The sequence FYXXINXG (FYING motif) in the 5th transmembrane domain (TM5) is invariably conserved among the members of the PTR (Peptide TRansport) family ranging from yeast to human. To test the role of TM5 in Ptr2p function, Ala-scanning mutagenesis of the 22 residues comprising TM5 was completed. All mutated transporters, with the exception of the Y248A mutant, were expressed as determined by immunoblots. In peptide-dependent growth assays, ten mutants of the non-FYING residues grew as well as wild-type Ptr2p on all twelve different peptides tested. All of the FYING motif mutants, except the non-expressed Y248A, plus seven other mutants in TM5 exhibited differential growth on peptides including Leu-Leu and Met-Met-Met indicating that these mutations conferred substrate preference. In assays measuring direct uptake of the radioactive peptides (3)H-Leu-Leu or (14)C-Met-Met-Met, the F, I and G mutants of the FYING motif did not demonstrate accumulation of these peptides over a ten minute interval. The mutation N252A of the FYING motif, along with L240A, M250A, and L258A, exhibited differential substrate preference for Met-Met-Met over Leu-Leu. Other mutations (T239A, Q241A, N242A, M245A, and A260) resulted in preference for Leu-Leu over Met-Met-Met. These data demonstrate that TM5, in particular its conserved FYING motif, is involved in substrate preference of Ptr2p.
Hauser
Melinda
M
Department of Microbiology, Walters Life Sciences, University of Tennessee, Knoxville, TN 37996, USA.
Kauffman
Sarah
S
Naider
Fred
F
Becker
Jeffrey M
JM
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D001692:Biological Transport / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D007930:Leucine / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D026901:Membrane Transport Proteins; C093495:PTR2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008715:Methionine; D007930:Leucine; D000409:Alanine