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. 2014 Nov;13(11):1380-92.
doi: 10.1128/EC.00094-14. Epub 2014 Aug 29.

Functional implications and ubiquitin-dependent degradation of the peptide transporter Ptr2 in Saccharomyces cerevisiae

Ken Kawai  1 , Atsuto Moriya  1 , Satoshi Uemura  1 , Fumiyoshi Abe  2
Affiliations

Functional implications and ubiquitin-dependent degradation of the peptide transporter Ptr2 in Saccharomyces cerevisiae

Ken Kawai et al. Eukaryot Cell. 2014 Nov.

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 PepTst 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 PepTst are involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enable ptr2Δtrp1 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.

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Figures

FIG 1
FIG 1
Multiple-sequence alignments of TMDs for some peptide transporters. The alignments were constructed for ScPtr2 (S. cerevisiae), CaPtr2 (C. albicans), hPepT1 and hPepT2 (H. sapiens), PepTso (S. oneidensis), PepTst (S. thermophilus), and GkPOT (G. kaustophilus) using the Praline multiple-sequence alignment program (54). TMD α-helices were assigned based on the crystallographic structure of PepTst (PDB 4APS) (28). Amino acid residues that mutated in this study are marked with an asterisk (*) above the residue, and those that reside in the extracellular gate in bacterial peptide transporters are marked with a dot (•).
FIG 2
FIG 2
Structural model of Ptr2. The structure of Ptr2 was based on the crystal structure of PepTst inward-open conformation (PDB 4APS) as a template (28). Amino acid residues that were mutated in this study are indicated.
FIG 3
FIG 3
Effects of Ptr2 mutations on cell growth. 3HA-tagged wild-type or Ptr2 mutant proteins were expressed in ptr2Δ cells. The cells were incubated at 25°C for 20 h in SD (40 μg · ml−1 tryptophan) (A), Ala-Trp medium containing 20 or 100 μg · ml−1 Ala-Trp in the absence of free tryptophan (B), or Leu-Trp medium containing 23 μg · ml−1 Leu-Trp in the absence of free tryptophan (C). The starting OD600 value was 0.04.
FIG 4
FIG 4
Expression of Ptr2 mutant proteins. 3HA-tagged wild-type or Ptr2 mutant proteins were expressed in ptr2Δ trp1 cells (A), PTR2 trp1 cells (B), or ptr2Δ TRP1 cells (C). Exponentially growing cells in SD medium were moved to Ala-Trp (AW) medium (20 μg · ml−1) and incubated for an additional 5 h. Whole-cell extracts were subjected to Western blot analysis to detect Ptr2-3HA and Adh1 using specific antibodies. Adh1 was used as a loading control.
FIG 5
FIG 5
Localization of Ptr2 mutant proteins in ptr2Δ cells. (A) GFP-tagged wild-type or Ptr2 mutant proteins were expressed in ptr2Δ cells. Exponentially growing cells in SD medium were moved to Ala-Trp (AW) medium (20 μg · ml−1) and incubated for an additional 5 h. The cells were imaged via a fluorescence microscope. Ptr2 in the plasma membrane is shown by arrowheads. Ptr2R93K-GFP and Ptr2W362L-GFP were predominantly localized in the vacuole, although they remained in the plasma membrane in some of the cells (∼25%, lower panels in R93K and W362L). DIC, differential interference contrast. (B) Whole-cell extracts were subjected to Western blot analysis to detect Ptr2-GFP and Adh1 using specific antibodies. Adh1 was used as a loading control.
FIG 6
FIG 6
Effects of Ptr2 mutations on Ala-Ala uptake. 3HA-tagged wild-type or Ptr2 mutant proteins were expressed in ptr2Δ cells. (A) Competition import assay for 3H-labeled Ala-Ala with nonlabeled Ala-Ala or Ala-Trp. The wild-type cells were incubated for 10 min with 3H-labeled Ala-Ala in the presence of 72.6 μM nonlabeled Ala-Ala (1×) or additional nonlabeled Ala-Ala or Ala-Trp. (B) Effects of Ptr2 mutations on Ala-Ala uptake. Cells were incubated in 3H-labeled Ala-Ala in the presence of 72.6 μM nonlabeled Ala-Ala. Data are expressed as mean values of amino acid incorporated (pmol · 107 cells−1 min−1) with standard deviations obtained from three independent experiments. Inset, time dependence of Ala-Ala uptake in the wild-type cells.
FIG 7
FIG 7
Ubiquitin-dependent degradation of Ptr2 proteins. (A) Rapid degradation of Ptr2 in the wild-type cells. (B) Stabilization of Ptr2 in the HPG1-1 (Rsp5P514T) and the bul1Δbul2Δ mutants cultured in SD medium. Whole-cell extracts were prepared after adding cycloheximide (CHX). Western blot analysis was performed to detect Ptr2-3HA and Adh1 using specific antibodies. Adh1 was used as a loading control.
FIG 8
FIG 8
Determination of lysine residues required for ubiquitin-dependent Ptr2 degradation. (A) Whole-cell extracts were prepared from ptr2Δ cells expressing Ptr2-3HA with single (top) or multiple (bottom) K-to-R (K>R) substitutions. SD medium was used for culture. Western blot analysis was performed to detect Ptr2-3HA and Adh1 using specific antibodies. Adh1 was used as a loading control. (B) The OD600 values were measured after individual strains were grown in Ala-Trp medium (20 μg · ml−1) at 25°C for 20 h. The starting OD600 value was 0.04. (C) GFP-tagged Ptr2 proteins with multiple K-to-R substitutions were expressed in ptr2Δ cells. Exponentially growing cells in SD medium were moved to Ala-Trp medium (20 μg · ml−1) and incubated for an additional 5 h. The cells were imaged using a fluorescence microscope. DIC, differential interference contrast.
FIG 9
FIG 9
Ubiquitin-dependent degradation of dysfunctional Ptr2 proteins. (A) 3HA-tagged wild-type or Ptr2 mutant proteins were expressed in ptr2Δ cells. Cycloheximide was added to the cells in SD medium for 2 h. (B and C) 3HA-tagged wild-type or Ptr2 mutant proteins were expressed in PTR2 or PTR2 HPG1-1 cells (B) and PTR2 or ptr2Δ HPG1-1 cells (C). (D) 3HA-tagged wild-type or Ptr2 mutant proteins combined with the 3K-to-R (3K>R) substitution were expressed in ptr2Δ cells. Exponentially growing cells in SD medium were moved to Ala-Trp (AW) medium (20 μg · ml−1) and incubated for an additional 5 h. Whole-cell extracts were subjected to Western blot analysis to detect Ptr2-3HA and Adh1 using specific antibodies. Adh1 was used as a loading control.
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References

    1. Steiner HY, Naider F, Becker JM. 1995. The PTR family: a new group of peptide transporters. Mol. Microbiol. 16:825–834. 10.1111/j.1365-2958.1995.tb02310.x. - DOI - PubMed
    1. Daniel H, Spanier B, Kottra G, Weitz D. 2006. From bacteria to man: archaic proton-dependent peptide transporters at work. Physiology (Bethesda) 21:93–102. 10.1152/physiol.00054.2005. - DOI - PubMed
    1. Newstead S. 2011. Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family. Biochem. Soc. Trans. 39:1353–1358. 10.1042/BST0391353. - DOI - PubMed
    1. Pao SS, Paulsen IT, Saier MH., Jr 1998. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 62:1–34. - PMC - PubMed
    1. Yan N. 2013. Structural advances for the major facilitator superfamily (MFS) transporters. Trends Biochem. Sci. 38:151–159. 10.1016/j.tibs.2013.01.003. - DOI - PubMed

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