Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.

Abstract

We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene.

Cloning of an Arabidopsis histidine transporting protein related to nitrate and peptide transporters

“…So far no data are available as to whether CHLl or the peptide transporters also transport histidine. Expression of these proteins in JT16 and expression of NTRl and CHLl in the yeast peptide transport mutant or expression in oocytes, as shown for the rabbit oligopeptide transporter, may be tools to study the inter-relationship of the transport activities of the three types of transporters [4,21,24]. Due to the comparable sequence similarities of the different transporters, the comparison did not help in defining the actual function.…”

“…Furthermore new genes could be identified that mediate growth of JT16 on media containing 6 mM histidine and that also encode integral membrane proteins. The gene described in this manuscript is a typical membrane protein with 12 putative membrane spanning regions and is homologous to two other types of transporters, namely the low affiity nitrate transporter from Arabidopsis and peptide transporters from yeast and mammalian origin [4,21,24].…”

“…A search through the databases shows 50-60% similarity to the Arabidopsis nitrate transporter (CHLl) and to the yeast (PTR2) and rabbit (PepTl) peptide transporters, but no homologies to the other Arabidopsis amino acid permeases [4,15,21,24]. A number of addiTo prove that NTRl is actually a plant gene, Southern blot hybridizations of Arabidopsis DNA were performed.…”

Cloning and functional characterisation of a peptide transporter expressed in the scutellum of barley grain during the early stages of germination

“…The isolation of a Saccharomyces cerevisiae peptide transport mutant (ptr2) facilitated the cloning of a yeast peptide transporter by functional complementation (Perry et al, 1994). Complementation of the ptr2 mutant with an Arabidopsis thaliana cDNA library subsequently resulted in the isolation of a root-specific di-and tripeptide transporter AtPTR2-A .…”

The Role of Small Wort Peptides in Brewing Fermentations

“…Initial peptide uptake rates were observed to be higher in cells that were grown on poor nitrogen sources, such as proline, than in cells grown on preferred nitrogenous materials, such as glutamine. Based on this criterion, it has been proposed that the peptide transport system of yeasts falls under the regulatory control of the nitrogen catabolite repression mechanism 18 . Probably a similar inactivation as happens in the GAP1 system, may take place for the peptide transport system, when yeast cells have been propagated in a medium with ammonium ions as the sole nitrogen source.…”