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Functional implications and ubiquitin-dependent degradation of the 1
peptide transporter Ptr2 in Saccharomyces cerevisiae 2
3
Ken Kawai, Atsuto Moriya, Satoshi Uemura and Fumiyoshi Abe* 4
5
Department of Chemistry and Biological Science, College of Science and Engineering, 6
Aoyama Gakuin University, Sagamihara, Japan 7
8
Keywords: Peptide transporter Ptr2, structural modeling, ubiquitination, Rsp5 ubiquitin ligase, 9
quality control 10
11
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*Corresponding author: Fumiyoshi Abe 13
5-10-1 Fuchinobe, Chuo-ku, Sagamihara 252-5258, Japan 14
Phone: +81-42-759-6233 15
Fax: +81-42-759-6511 16
E-mail: [email protected] 17
18
19
EC Accepts, published online ahead of print on 29 August 2014Eukaryotic Cell doi:10.1128/EC.00094-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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ABSTRACT 20
The peptide transporter Ptr2 plays a central role in di- or tripeptide import in Saccharomyces 21
cerevisiae. Although PTR2 transcription has been extensively analyzed in terms of 22
upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this 23
transporter are limited. Here we identified 14 amino acid residues required for peptide import 24
through Ptr2 based on the crystallographic information of Streptococcus thermophiles peptide 25
transporter PepTst, and based on the conservation of primary sequences among the 26
proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 27
mutations of which the corresponding residues of PepTst are involved in peptide recognition, 28
salt-bridge interaction, or peptide translocation failed to enable ptr2trp1 cell growth in 29
alanyl–tryptophan (Ala–Trp) medium. We observed that Ptr2 underwent rapid degradation 30
after cycloheximide treatment (half-life approximately 1 h), and this degradation depended on 31
Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 32
16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 33
degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, 34
W362L, and E480D) exhibited severe defects in peptide import and were subjected to 35
Rsp5-dependent degradation when cells were moved to Ala–Trp medium, whereas, similar to 36
the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results 37
suggest that Ptr2 undergoes quality control, and the defects in peptide binding and the 38
concomitant conformational change render Ptr2 for efficient ubiquitination and subsequent 39
degradation. 40
41
INTRODUCTION 42
Nitrogen is imported into the cell in the form of an ammonium ion, amino acid, or peptide 43
through transporter proteins in the plasma membrane. Peptide transporters typically comprise 44
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12 transmembrane domains (TMDs) that are highly conserved among bacteria, animals, and 45
plants. These transporters belong to the proton-coupled oligopeptide transporter (POT) family, 46
which is also referred to as the peptide transporter (PTR) family (1–3). The POT family 47
belongs to the major facilitator superfamily (MFS), which contain substrate/H+
symporters 48
that are driven by electrochemical gradient across the plasma membrane (4, 5). In addition to 49
being a source of nitrogen, peptides play beneficial roles in physiological activity. For 50
example, soy peptides, the proteolytic products of soy proteins, can reduce serum lipid levels, 51
scavenge free radicals, and reduce oxidative stress in mammalian cells (6–8). In yeast, soy 52
peptides improve tolerance to freeze-thaw stress, repress the formation of lipid bodies (9), and 53
accelerate ethanol fermentation (10, 11). hPepT1 and hPepT2 are human peptide transporters 54
expressed in the small intestine and the kidney, respectively (12). hPepT1 operates as a 55
high-capacity low-affinity peptide transporter, whereas hPepT2 operates as a low-capacity 56
high-affinity transporter. In addition to peptide uptake, these transporters mediate absorption 57
of drugs that have a steric resemblance to peptides, such as -lactam antibiotics, 58
anti-hypertensive drugs, anti-cancer agents, and antivirals (e.g., valacylovir) (13–17). Hence, 59
investigation of POT family proteins should facilitate fermented food production and drug 60
discovery, as well as contribute to our basic understanding of nutrient transport. 61
In the yeast Saccharomyces cerevisiae, the POT/PTR family protein Ptr2 is the major 62
peptide transporter for importing di- and tripeptides (1, 18, 19). External amino acids regulate 63
PTR2 transcription through the Ssy1-Ptr3-Ssy5 (SPS) signal transduction pathway. In the SPS 64
complex, Ssy1 is a plasma membrane protein that senses external amino acids and induces 65
transcription of PTR2 and several amino acid permease genes (20-23). Dipeptides containing 66
N-terminal basic (Arg, Lys, and His) or bulky hydrophobic (Phe, Leu, Tyr, Trp, and Ile) amino 67
acids, the N-end rule amino acids, also positively regulate PTR2 transcription (24, 25). These 68
dipeptides bind directly to Ubr1 (also known as Ptr1), which leads to Ubr1-dependent 69
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degradation of Cup9, a repressor of PTR2 transcription, and consequently, PTR2 upregulation 70
(24, 25). Although PTR2 transcription has been extensively investigated, the mechanism by 71
which the Ptr2 protein recognizes and imports peptides is still unclear. To the best of our 72
knowledge, the only relevant study is a mutational analysis on TMD5 of Ptr2 (26). The 73
FVxxINxG sequence in TMD5 is highly conserved among the POT/PTR family proteins, 74
from yeast to human. Although substitutions of F244, F247, Y248, I251, or G254 to alanine 75
abolished the uptake of Leu–Leu and Met–Met–Met, N252A substitution exhibited 76
differential substrate preference for Met–Met–Met over Leu–Leu (26). Thus, at least TMD5 77
plays a role in Ptr2 substrate recognition. 78
Recently, crystal structures of bacterial homologs for mammalian peptide transporters 79
were solved in PepTso from Shewanella oneidensis (27), PepTst from Streptococcus 80
thermophilus (28) and GkPOT from Geobacillus kaustophilus (29). These crystal structures 81
share a common fold in the MFS proteins consisting of two 6-transmembrane bundles facing 82
together in the membrane with a central cavity between the two bundles. The PepTso structure 83
formed a substrate-bound occluded state (27), whereas the PepTst structure formed an 84
inward-open conformation (28). The GkPOT structures were both an inward-open H+-bound 85
and an inward-open substrate/H+-bound conformations (29). These structures revealed the key 86
mechanistic and functional roles for conserved glutamate (or aspartate) and arginine residues 87
in the peptide-binding site, as well as a number of amino acid residues involved in peptide 88
recognition. Interestingly, both glutamate and arginine residues are not conserved in fungal 89
Ptr2, including S. cerevisiae and Candida albicans in which glycine and serine residues are 90
positioned, respectively. This suggests an unidentified mechanism of recognition or 91
translocation of substrates in fungal Ptr2 proteins, which might account for the unsolved 92
effects of soy peptides in yeast physiology. 93
Homology modeling that uses the crystal structure of a closely related protein as a 94
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template allows us to determine the functional roles of corresponding amino acid residues in 95
structurally unknown proteins. In this study, we introduced mutations into Ptr2 to verify the 96
significance of 19 amino acid residues based on the structural information of the POT family 97
proteins. Yeast genetics has an advantage in that a functional analysis of a transporter protein 98
can be performed in vivo in combination with an analysis of the expression and subcellular 99
localization of the protein. We also show that Ptr2 undergoes Rsp5-dependent ubiquitination 100
that likely occurs at N-terminal lysine positions at 16, 27, and 34. 101
102
MATERIALS AND METHODS 103
Yeast strains and culture conditions 104
The wild-type strain YPH499 (MATa his3-Δ200 leu2-Δ1 lys2-801 trp1-Δ1 ade2-101 ura3-52), 105
the ptr2Δ mutant (ptr2Δ::KanMX in YPH499, see below), and the ptr2ΔTRP1 mutant 106
(ptr2Δ::KanMX TRP1 in YPH499, see below) were used in this study. Unless otherwise 107
specified, ptr2Δ denotes the ptr2Δ (trp1) mutant. Cells were grown in synthetic dextrose (SD) 108
medium or L-alanyl-L-tryptophan (Ala–Trp) medium at 25 °C with shaking. Ala–Trp medium 109
was supplemented with Ala–Trp to complement tryptophan auxotrophy (trp1) instead of 110
L-tryptophan in SD medium, and was used for functional analysis of ptr2 mutants. Ala–Trp 111
medium was composed of yeast nitrogen base without amino acids (6.7 g•L−1), D-glucose (20 112
g•L−1), adenine (20 µg•mL−1), histidine (20 µg•mL−1), lysine (30 µg•mL−1), leucine (90 113
µg•mL−1), and Ala–Trp (20 µg•mL−1). In addition, L-leucyl-L-tryptophan (Leu–Trp) was 114
used to complement tryptophan auxotrophy. Ala–Trp, Leu–Trp, L-alanyl–L-leucine (Ala–Leu), 115
and L-alanyl–L-alanine (Ala–Ala) were purchased from Kokusan Chemical Co. Ltd. (Tokyo, 116
Japan). 117
118
Construction of the ptr2Δ mutant and plasmids 119
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PCR-based gene disruption was employed in YPH499 to create the ptr2Δ::KanMX mutant 120
using the genome DNA from the ptr2Δ mutant (ptr2Δ::KanMX in BY4742) as a template, and 121
the primers 5’-GGTTTGTAAAATATATAAGTGATGTACA-3’ and 122
5’-CATTTGAGATAATGGTATGCAAATACGC-3’. The transformant was selected on YPD 123
agar plates containing 200 µg•mL−1
geneticin. pJJ280 (TRP1) (30) was digested with EcoRI 124
to obtain a TRP1 DNA fragment, which was subsequently introduced into the ptr2Δ mutant to 125
generate the ptr2Δ TRP1 mutant. 126
The plasmids used in this study are listed in Table 1. pUA35 (TDH3 promoter 127
[pTDH3]-3HA-TDH3 terminator [tTDH3], URA3 CEN4) was used to generate a PTR2-3HA 128
plasmid driven by its own promoter. pUA35 was digested with XhoI and SmaI to remove 129
pTDH3. The PTR2 ORF, with its own promoter region (800 bp upstream of the ORF), was 130
amplified by PCR using the genomic DNA from strain YPH499 as a template and 131
5’-CGGGCCCCCCCTCGAGGAAGGCAGCGGCGAACTGACTT-3’ and 132
5’-ACTCATGGTTCCCCCGGGATATTTGGTGGTGGATCTTAGACTTTCCATTG-3’ as the 133
primers. The digested plasmid YCplac33 (URA3 CEN4) (31) and the PCR product were fused 134
by In-Fusion HD Cloning Kit (TaKaRa Bio Inc., Otsu, Shiga, Japan) to yield pPTR2-3HA 135
(URA3 CEN4). PCR-based site-directed mutagenesis was performed to create each of the 19 136
amino acid substitutions in Ptr2 TMDs or at least one lysine-to-arginine (K > R) substitution 137
in the Ptr2 N-terminal domain using pPTR2-3HA as a template with the corresponding mutant 138
primers. To generate a PTR2-GFP plasmid, pPTR2-3HA was digested with XhoI and SmaI to 139
obtain a DNA fragment containing the PTR2 ORF with its own promoter region. The resulting 140
fragment was cloned into pKK1 (GFP URA3 CEN4) to yield pPTR2-GFP. 141
142
Functional analysis of Ptr2 mutants 143
The wild-type or ptr2 mutant cells were grown in SD medium at the OD600 value up to about 144
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1.0 (1.65 × 107 cells •mL
−1). The cells were collected by centrifugation, washed twice with 145
distilled water, and resuspended in SD or Ala–Trp medium to obtain cultures at the OD600 146
value of 0.04. The OD600 values of cultures were measured after incubating for 20 h at 25 °C. 147
148
Western blot analysis 149
Whole cell extracts were prepared as previously described (32). In brief, 108 cells were 150
collected by centrifugation, washed once with 10 mM NaN3-10 mM NaF, and washed once in 151
lysis buffer A (50 mM Tris-HCl, 5 mM EDTA, 10 mM NaN3, 1 mM phenylmethylsulfonyl 152
fluoride, and one tablet of EDTA-free Complete [Roche, Mannheim, Germany] for 50 mL 153
[pH 7.5]). The cells were broken with glass beads. Unbroken cells and debris were removed 154
by centrifugation at 900 g for 5 min, and the cleared lysates were treated with 5% SDS and 155
5% 2-mercaptoethanol at 37 °C for 10 min to denature proteins. Western blot analysis was 156
performed as previously described (32) using antibodies for HA (16B12, BabCO, Richmond, 157
CA, USA), GFP (anti-chicken IgY, Aves Labs, Inc., Tigard, OR, USA) and Adh1 (Rockland 158
antibodies and assays, Gilbersville, PA, USA). Adh1 was used as a loading control for the 159
whole cell extracts. An image analyzer (LAS4000 mini; GE Healthcare Life Sciences, 160
Piscataway, NJ, US) was used for signal detection. 161
162
Fluorescence microscopy 163
Living cells expressing GFP-tagged Ptr2 proteins were placed on a glass slide and were 164
imaged using a fluorescence microscope model IX70 with 100× magnification of the 165
objective lens (Olympus, Co. Ltd, Tokyo, Japan). 166
167
Peptide import assay 168
Peptide import was analyzed using the procedure for tryptophan import by Tat2, but with 169
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modifications (33). A commercially available radiolabeled peptide, [3H] L-alanyl-L-alanine 170
(Ala–Ala, MT-945, 14.8 GBq•mmol−1
, Moravek Biochemicals, Inc., Brea, CA, USA), was 171
used for the peptide import assay. Cells were grown in SD medium at a cell density of up to 172
1.5 × 107
cells•mL−1
. The cells were collected by centrifugation, washed twice, and 173
resuspended in SD medium at a cell density of about 5.0 × 107
cells•mL−1
. The import assay 174
was performed in the presence of 11.6 µg•mL−1
of nonlabeled Ala–Ala with a 1/2000 volume 175
of the 3H-labeled Ala–Ala. The molar concentration of nonlabeled Ala–Ala (72.6 µM) was 176
equal to that for the 20 µg•mL−1
Ala–Trp used in the functional analysis. After incubation, the 177
cells were trapped with a vacuum aspirator on GF/C glass filters that were stacked in pairs. 178
The filters were washed with 10 mL cold distilled water. The quantity of imported peptides 179
was measured using a liquid scintillation counter. In our experimental condition, 1 DPM of 180
[3H] Ala–Ala can be converted to 42.4 fmol Ala–Ala. Data were expressed as mean values of 181
Ala–Ala incorporated (pmol•107
cells−1•min
−1) with standard deviations obtained from three 182
independent experiments. 183
184
Structural modeling 185
The ESyPred3D Web Server 1.0 software 186
(http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/) was used for modeling, an 187
automated homology modeling program with an increased alignment performance that is 188
based on the modeling package MODELLER (34,35). The profile query for Ptr2 yielded the S. 189
thermophilus PepTst (PDB entry: 4APS) as a structural template (28). An alignment was 190
created for the template, and the Ptr2 model was constructed using the PyMOL Molecular 191
Graphics System, Version 1.5.0.4 Schrödinger, LLC (http://www.pymol.org/) (36). 192
193
RESULTS 194
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Rational for mutation design and homology modeling of Ptr2 195
Figure 1 shows a multiple sequence alignment of TMDs in some peptide transporters. The 196
ExxERFxYY motif in TMD1 is highly conserved across the POT family proteins and 197
contributes to forming the central cavity within the molecule (3). In PepTst, E25 and R26 (E92 198
and R93 in Ptr2, respectively) interact through a salt bridge, which is positioned close to E22 199
(E89 in Ptr2) in TMD1. E22Q, E25Q, and R26K mutations completely inactivate 200
proton-driven peptide uptake but retain some activity of counterflow in PepTst (28). Y29 201
(TMD1) and Y68 (TMD2) (Y96 and Y138 in Ptr2, respectively) are thought to help determine 202
peptide specificity. K126 in TMD4 (K205 in Ptr2) is conserved throughout the POT family 203
proteins and is positioned close to the ExxERFxYY motif. K126 and E400 (TMD10) are 204
positioned to form the peptide-binding site. The K126A mutation only abolishes 205
proton-driven peptide import and retains counterflow activity, whereas E400D mutation 206
abolishes both functions (28). In human hPepT1, the equivalent glutamate (E595) is assumed 207
to bind the N-terminus of peptides. W294 in hPepT1 (W362 in Ptr2) plays a role in peptide 208
import (37). Hence, we created E92Q, R93K, Y96A, Y138A, K205R, W362L, and E480D 209
mutations in Ptr2. S130 (TMD4), F148 (TMD5), G407 (TMD10), L408 (TMD10), M424 210
(TMD11), and W427 (TMD11) in PepTst (S209, F244, G487, L488, M504, and F507 in Ptr2, 211
respectively) are positioned at the intracellular gate, which shows hinge-like movement. 212
Hence, we created S209A, F244L, G487A, L488A, M504L, and F507W mutations in Ptr2. In 213
addition to the potentially important 13 amino acid residues, we mutated the following 6 sites 214
according to the pronounced conservation: D149E (TMD2), G168A (TMD3), L196A (TMD4), 215
F247L (TMD5), T510V (TMD11), and G514A (TMD11). R33 and E300 in PepTst are highly 216
conserved across the POT family, and the salt bridge between R33 and E300 is assumed to 217
facilitate close packing in the extracellular gate between TMD1–2 and TMD7–8 (28). In total, 218
19 Ptr2 mutants were subjected to the subsequent functional analysis. 219
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For the structural modeling of Ptr2, the ESyPred3D Web Server 1.0 software proposed an 220
inward-open crystal structure of PepTst (PDB: 4APS) as the closest template (28). In the 221
overall modeled structure, the N- and C-terminal 6-helix bundles TMD1–6 and TMD7–12 222
face each other to form a pseudo two-fold symmetry structure (Fig. 2). There seems to be a 223
putative trajectory for a substrate peptide in the top view of the modeled Ptr2. The 19 amino 224
acid residues mutated in this study are mapped in the structure (Fig. 2). 225
226
Functional analysis of Ptr2 mutant proteins 227
To evaluate the activity of Ptr2 mutant proteins in vivo, we performed a growth assay using 228
nutrient auxotrophy for tryptophan and leucine. First, we examined whether Ala–Trp or Ala–229
Leu supported growth of the wild-type or ptr2 mutant in the absence of tryptophan or 230
leucine in SD medium, respectively. We confirmed the wild-type strain grew on 20 µg•mL−1
231
Ala–Trp and 40 µg•mL−1
tryptophan with equal efficiency, but ptr2 mutant growth was 232
defective in 20 µg•mL−1
Ala–Trp (Fig. 3A). Ala–Leu did not support wild-type growth, even 233
with a very high concentration of 100 µg•mL −1
, suggesting that the peptide form of leucine 234
cannot meet its high demand in protein synthesis (data not shown). Therefore, Ala–Trp was 235
used for further experiments. Hereafter, the wild-type strain denotes the ptr2 cell harboring 236
pPTR2-3HA, and the ptr2 mutant denotes the cell harboring an empty vector alone. 237
Cells were grown in SD medium and diluted with Ala–Trp medium to obtain cultures of 238
0.04 OD600. After incubating for 20 h at 25 °C, the OD600 values were measured to quantify 239
Ptr2 mutant growth. The 19 Ptr2 mutant cells exhibited growth in the SD medium that was 240
equal to that of the wild-type cells, whereas the mutants showed varied degrees of deficient 241
growth in Ala–Trp medium (Fig. 3B). E92Q, R93K, Y138A, K205R, F244L, W362L, E480D, 242
M504L, F507W, and T510V mutants showed severe growth defects. Y96A, F247L, G487A, 243
and L488A mutants showed moderate growth defects, which were partially repressed by a 244
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higher concentration of Ala–Trp (100 µg•mL−1
). In contrast, D149E, G168A, L196A, S209A, 245
and G514A mutations conferred no obvious growth defect in Ala–Trp (20 µg•mL−1
) medium 246
(Fig. 3B). A similar result was obtained with a medium containing an N-end rule dipeptide 247
Leu–Trp (23 µg•mL−1
), which was preferred by Ptr2 (25), except for the marked growth 248
defect in the G514A mutant (Fig. 3C). 249
250
Expression and localization of Ptr2 mutant proteins 251
Next, we examined whether the mutations affected Ptr2 expression. Cells were grown in SD 252
medium and then moved to Ala–Trp (20 µg•mL−1
) medium to continue incubating for 5 h. 253
Whole cell lysates were prepared for Western blot analysis to detect Ptr2-3HA proteins. 254
Consistent with previous reports (25), Ptr2 was upregulated after cells were moved to Ala–Trp 255
medium (Fig. 4A). The Ptr2 mutant proteins were also expressed in SD medium, though the 256
expression levels varied among the strains. We found that Ptr2 carrying the E92Q, R93K, 257
K205R, W362L, or E480D mutation was markedly downregulated after moving the cells to 258
Ala–Trp medium. In contrast, the remaining Ptr2 mutant proteins were upregulated as they 259
were in the wild-type Ptr2 (Fig. 4A). It should be noted that E92, R93, K205, W362, and 260
E480 are assumed to form the substrate-binding pocket in the Ptr2 structural model (Table 2 261
and Fig. 2), thereby raising the possibility that the 5 substrate-free forms of Ptr2 may have 262
been efficiently degraded when cells were cultured in Ala–Trp medium. When Ptr2 proteins 263
were expressed in the wild-type PTR2 trp1 strain, the 5 Ptr2 mutant proteins were upregulated 264
when moved to Ala–Trp medium, similar to that observed in the wild-type Ptr2 (Fig. 4B). 265
Therefore, intracellular Ala–Trp may have prevented the 5 Ptr2 mutant proteins from 266
degradation in the wild-type cells. Further, we examined whether conferring tryptophan 267
prototrophy in the ptr2 strain, which enabled growth in Ala–Trp medium, affected the 268
degradation of the 5 Ptr2 mutant proteins. We found that the 5 Ptr2 mutant proteins were 269
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degraded when the ptr2 TRP1 cells were moved to Ala–Trp medium; however, the 270
degradation of Ptr2 was delayed (Fig. 4C). These results suggest that the substrate-binding 271
defect of Ptr2 and the shortage of intracellular tryptophan promoted Ptr2 degradation (see 272
Discussion). 273
GFP-tagged mutant proteins were expressed in ptr2 cells to determine the extent to 274
which the mutations affected Ptr2 localization. The wild-type Ptr2-GFP was localized to the 275
plasma membrane when cells were incubated in both SD and Ala–Trp medium (Fig. 5A, 276
arrowhead). Intensive GFP fluorescence in the vacuole suggests that Ptr2 is subjected to 277
degradation through the endocytic pathway. We confirmed that all mutant Ptr2-GFP proteins 278
were localized to the plasma membrane in cells grown in SD medium, and the vacuoles were 279
intensively stained with GFP (Fig. 5A, lower panels in R93K and W362L). In fact, partially 280
digested products of Ptr2-GFP, which were attributed to the vacuolar signal, were observed in 281
Western blotting using an anti-GFP antibody (Fig. 5B). Therefore, the mutant Ptr2 proteins 282
can be delivered to the cell surface as efficient as the wild-type protein, and are delivered to 283
the vacuole for degradation. Consistent with the Western blotting results in Fig. 4, Ptr2-GFP 284
protein carrying the E92Q, K205R, or E480D mutation was absent from the plasma 285
membrane when moved to Ala–Trp medium (Fig. 5A). Ptr2-GFP proteins carrying the R93K 286
or W362L mutation were predominantly localized in the vacuole when moved to Ala–Trp 287
medium, although these remained in the plasma membrane in some of the cells (~25%) (Fig. 288
5A). In addition, Ptr2R93K
-GFP and Ptr2W362L
-GFP were not efficiently degraded when moved 289
to Ala–Trp medium (Fig. 5B); however, Ptr2R93K
-3HA and Ptr2W362L
-3HA were rapidly 290
degraded (Fig. 4A). These findings suggest that the C-terminal fused GFP partially stabilizes 291
Ptr2R93K
-GFP and Ptr2W362L
-GFP in the plasma membrane. In addition, we expressed 292
GFP-tagged mutant Ptr2 in the wild-type PTR2 cells and observed that they were localized to 293
the plasma membrane, similar to the wild-type Ptr2 protein (data not shown). For the 294
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subsequent peptide import assay to be valid, we used SD medium rather than Ala–Trp 295
medium for cell culture to ensure plasma membrane localization of Ptr2. 296
297
Peptide uptake by Ptr2 mutant proteins 298
To evaluate the effect of Ptr2 mutations quantitatively, we employed peptide import assay 299
using a commercially available [3H] Ala–Ala. First, we performed a competitive assay to 300
examine whether the use of Ala–Ala was valid and consistent with the functional analysis 301
with Ala–Trp. Exponentially growing cells in SD medium were collected, washed, and 302
transferred to [3H] Ala–Ala medium containing 11.6 g•mL
–1 nonlabeled Ala–Ala with a 303
molar concentration that was equal to that for 20 g•mL–1
Ala–Trp (72.6 M). The assay was 304
performed in the presence or absence of nonlabeled Ala–Ala or Ala–Trp in varying 305
concentrations as competitive substrates. We found that [3H] Ala–Ala uptake decreased when 306
nonlabeled Ala–Ala increased, but was more effective with nonlabeled Ala–Trp (Fig. 6A). 307
This finding suggests that both peptides can be transported by Ptr2, but Ala–Trp is preferred 308
over Ala–Ala. Such a preference is consistent with a recent report of a systematic competitive 309
assay that used the fluorescence tracer substrate -Ala–Lys (AMCA) (38). 310
Eight Ptr2 mutants, E92Q, R93K, Y138A, K205R, W362L, E480D, F507W, and T510V 311
were selected for peptide import assay, according to the extent of their growth defects. Peptide 312
uptake by the wild-type cells was time-dependent and exhibited a linear dependence on 313
incubation time up to 5 min (Fig. 6B inset). Due to the technical difficulties to analyze 10 314
samples simultaneously, subsequent measurements were performed at 7.5 min. The wild-type 315
cells imported Ala–Ala at 61 pmol•107 cells
−1•min−1
, whereas the ptr2 cells did not 316
imported it, confirming the primary role of Ptr2 in peptide import (Fig. 6B). Although the 317
experimental conditions differed, the degree of Ptr2-mediated Ala–Ala uptake was 20-fold 318
greater than Tat1-mediated tyrosine uptake and 40-fold greater than Tat2-mediated tryptophan 319
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uptake in which the import assay was performed on 19.6 M of tyrosine and tryptophan, 320
respectively (33). At 15-min incubation, a trace amount of Ala–Ala uptake (0.5 pmol•107 321
cells−1•min
−1) was observed in the ptr2 mutant. This could be mediated by another peptide 322
transporter, which is likely to be the allantoin transporter Dal5 because Opt1 and Opt2 cannot 323
import di/tripeptides (39). We found that all 8 Ptr2 mutants showed defective peptide uptake 324
with rates up to 4.2 pmol•107 cells
−1•min−1
(Fig. 6B). Accordingly, the 8 amino acid residues 325
are important for peptide recognition and translocation through the Ptr2 molecule. 326
327
Ptr2 undergoes ubiquitin-dependent degradation 328
To the best of our knowledge, Ptr2 has not been examined for ubiquitin-dependent 329
degradation. Therefore, we examined whether degradation of Ptr2 and its mutant forms were 330
dependent on Rsp5 ubiquitin ligase. We found that the wild-type Ptr2 was degraded rapidly in 331
cells cultured in both SD and Ala–Trp medium with the addition of cycloheximide (half-life 332
approximately 1 h), suggesting a high basal turnover for Ptr2 (Fig. 7A). The HPG1-1 mutant 333
is defective in Rsp5 ubiquitin ligase activity due to an amino acid substitution (P514T) in the 334
catalytic HECT domain of Rsp5 (32). Double deletion of genes encoding Rsp5-binding 335
proteins Bul1 and Bul2 also compromises Rsp5 activity upon ubiquitination (32). We found 336
that degradation of Ptr2 was significantly delayed in the HPG1-1 and the bul1bul2 mutants 337
compared with that in the wild-type strain, suggesting that Ptr2 undergoes Rsp5-mediated 338
ubiquitination (Fig. 7B). Covalent binding of ubiquitin to lysine residues likely occurs in the 339
cytoplasmic N-terminal domain in amino acid permeases including Gap1 (40), Tat1 (41), Tat2 340
(42), and Bap2 (43). Based on homology modeling by PepTst (PDB entry: 4APS) as a 341
structural template (Fig. 2) (28), the TMD1 of Ptr2 starts with isoleucine at position 78, and 342
thereby the cytoplasmic N-terminal domain is likely composed of 77 amino acids in which 4 343
lysine residues are located. We first created single K > R substitutions in the N-terminal 344
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domain to determine whether lysine is required for Ptr2 degradation. Whole cell extracts were 345
prepared from ptr2 cells expressing 3HA-tagged Ptr2 variant proteins in SD medium. We 346
found that K34R mutation resulted in retarded Ptr2 degradation after 2 h of cycloheximide 347
treatment (Fig. 8A). This stabilizing effect by the K34R mutation was more prominent after 1 348
h of cycloheximide treatment with respect to degradation of the wild-type Ptr2 (Fig. 8A). 349
These results suggest that K34 is primarily ubiquitinated by Rsp5 for degradation. Similar 350
results were obtained with cell culture in Ala–Trp medium (data not shown). The 4 single K > 351
R substitutions conferred normal growth on ptr2cells in Ala–Trp medium, which suggests 352
the K > R variants are functional (Fig. 8B). To examine whether the remaining lysines are 353
required for Ptr2 degradation at its maximum rate, we created multiple K > R substitutions in 354
combination with K34R. Compared with the single K34R mutation, double mutations 355
K16R-K34R and K27R-K34R further stabilized Ptr2, which was more pronounced in triple 356
(K16R-K27R-K34R; 3K > R) and quadruple (K16R-K27R-K34R-K77R; 4K > R) mutations 357
(Fig. 8A). This result suggests that multiple N-terminal lysines are potential targets for 358
Rsp5-mediated ubiquitination. As observed in the ubiquitination-deficient plasma membrane 359
proteins, Ptr23K>R
-GFP and Ptr24K>R
-GFP were predominantly localized to the plasma 360
membrane in cells in both SD and Ala–Trp medium in the absence of vacuolar GFP signals 361
(Fig. 8C). Interestingly, ptr2cells expressing the 3K > R or 4K > R mutant Ptr2 grew in 362
Ala–Trp medium less efficiently than cells expressing the wild-type Ptr2 or double K > R 363
mutants (Fig. 8B). We speculate the cells consumed Ala–Trp in excess as a result of Ptr23K>R
364
or Ptr24K>R
accumulation, and hence, Ala–Trp became insufficient for supporting full growth 365
of the Trp– strain. Otherwise, excess cytoplasmic Ala–Trp might abrogate amino acid 366
homeostasis in the cell. 367
Next, we examined whether peptide-induced degradation of the 5 dysfunctional Ptr2 368
mutants E92Q, R93K, K205R, W362L, and E480D (Fig. 4A) was also ubiquitin dependent. 369
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Initially, there was no measurable difference in steady-state turnover between the wild-type 370
Ptr2 and the dysfunctional Ptr2 mutants on treatment with cycloheximide (Fig. 9A). The 371
HPG1-1 cells expressing Ptr2 with each mutation were grown in SD medium and then moved 372
to Ala–Trp medium to culture for 5 h. We found that the 5 Ptr2 mutant proteins were 373
markedly stabilized in the PTR2 HPG1-1 mutant and the ptr2HPG1-1 mutant in Ala–Trp 374
medium, suggesting that Rsp5 mediates degradation of dysfunctional Ptr2 (Fig. 9B, C). In 375
support of this finding, the introduction of the 3K > R mutation to the 5 Ptr2 mutants 376
markedly stabilized the proteins (Fig. 9D). We assume that Ptr2 is subjected to 377
Rsp5-dependent quality control when the peptide import activity is severely impaired. 378
Because the 5 amino acid residues are assumed to reside in the peptide-binding pocket, these 379
Ptr2 mutants might fail to bind peptides thus preventing concomitant conformational change. 380
Therefore, peptide-free outward-facing conformation of Ptr2 might be the substrate for Rsp5 381
ubiquitin ligase. 382
383
DISCUSSION 384
Structural and functional implications of Ptr2 385
In this study, we identified amino acid residues required for Ptr2-mediated peptide import 386
using site-directed mutagenesis with the crystal structure of the S. thermophilus PepTst. The 387
growth assay of Ptr2 mutants with 20 µg•mL−1
Ala–Trp as a tryptophan source allowed us to 388
determine dysfunctional Ptr2 mutations when combined with the ptr2trp1 genetic 389
background. Of the 19 amino acid residues tested, 14 contributed to peptide import to various 390
degrees, however, the remaining 5 residues were not although they are highly conserved 391
across the POT family proteins. The 14 mutations are likely to cause defects in peptide 392
recognition or translocation through the Ptr2 molecule, but not defects in structural stability or 393
delivery to the plasma membrane of this protein. Inevitably, amino acid residues assumed to 394
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participate in proton-driven peptide uptake (E92 and R93), determining peptide specificity 395
(Y96 and Y138), peptide binding (K205, E480 and W362), and hinge-like movement at the 396
intracellular gate (F244, G487, L488, M504 and F507) play important roles in Ptr2 function. 397
This suggests that the mechanisms for peptide recognition and translocation are similar 398
between Ptr2 and other POT family proteins. However, Ptr2 and PepTst showed different salt 399
bridge interactions at the extracellular gate. In PepTst, two salt bridge interactions facilitated 400
close packing of the extracellular gate helices, which were R53 with E312 and R33 with E300 401
(28). These amino acid residues are not conserved in fungal peptide transporters (S123 with 402
M378 and S100 with G366, respectively). Mutational analysis in PepTst showed that the R53–403
E312 salt bridge plays a supportive role, whereas the R33–E300 salt bridge is essential during 404
peptide transport. E300A mutation abolishes transport in both proton-driven and 405
peptide-driven counterflow assay of PepTst, whereas R33A mutation abolishes transport only 406
in proton-driven assay (28). These interactions are considered important for orchestrating 407
alternating access with the POT family proteins. Therefore, salt bridges other than those 408
corresponding to PepTst R33–E300 might play a role at the putative extracellular gate in Ptr2. 409
Otherwise, the extracellular gate might be unnecessary in fugal peptide transporters. Currently, 410
Ptr2 has not been sufficiently studied for proton/peptide coupling transport and counterflow 411
activity. Such an analysis in combination with mutational mapping is crucial for describing 412
the undefined features of Ptr2 in peptide import. 413
One of the central issues to be solved through peptide transporter research is how a single 414
transporter can recognize many di/tripeptides for transport. The sequence FYxxINxG in 415
TMD5 is highly conserved in the POT family proteins. Alanine-scanning mutagenesis 416
performed on the 22 amino acid residues in TMD5 revealed that the FYxxINxG sequence 417
plays a role in substrate preference: the wild-type Ptr2 prefers Leu–Leu to Met–Met–Met for 418
uptake at the ratio of 0.6. Five mutations T239A, Q241A, N242A, M245A, and A260G 419
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resulted in an increased preference for Leu–Leu over Met–Met–Met relative to the wild-type 420
preference, whereas 4 mutations, L240A, M250A, N252A, and L258A, conferred a 421
preference for Met–Met–Met rather than Leu–Leu, particularly N252A and L258A (19). It is 422
rationalized in the modeled structure of Ptr2 that most of the 9 residues are facing toward the 423
putative peptide-binding site. The substrate multispecificity of Ptr2 was examined recently 424
using a dipeptide library in which dipeptides composed of hydrophobic or bulky amino acids 425
(Trp, Phe, His, Tyr, Met, or Leu) were preferred by Ptr2, whereas negatively charged and/or 426
small amino acids (Asp, Glu, Gly, or Pro) were not (38). Ala–Trp was categorized into the 427
high-affinity preferable substrate. Transcription of PTR2 is regulated by the N-end rule 428
pathway, through which dipeptide binding with a certain N-terminus to Ubr1 promotes 429
degradation of Cup9, a repressor of PTR2 (24,25). Consistently, the N-terminus of peptides 430
with a high-affinity for Ptr2 mostly comprises the N-end rule amino acids (Trp, Phe, His, Tyr, 431
Leu, Ile, and Lys)(38). In a Ptr2 structural model based on the crystal structure of S. 432
oneidensis PepTso (PDB 2XUT), a dipeptide is situated in a peptide-binding pocket that is 433
surrounded by multiple amino acid residues including R93, Y138, K205, and E480 Most 434
recently, there was one substantive publication on structural issues with the substrate 435
multispecificity of PepTst (44). The study presented crystal structures of di- and 436
tripeptide-bound complexes of PepTst, which revealed that Ala–Phe was oriented laterally in 437
the binding site, whereas Ala–Ala–Ala showed an alternative vertical binding mode. 438
Furthermore, the peptide-biding site can co-opt the same or similar pockets to accommodate 439
the side chains of different ligands, linking their formation and dissolution with the different 440
states in the transport cycle to facilitate peptide recognition and release. 441
442
Ubiquitin-dependent degradation of Ptr2 443
PTR2 has been analyzed extensively with respect to transcriptional regulation in S. cerevisiae 444
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(45-47) and Schizosaccharomyces pombe (48). Peptide import is upregulated by growth media 445
containing poor nitrogen sources such as allantoin, isoleucine, or proline due to upregulation 446
of PTR2 transcription (49). In media containing rich nitrogen sources such as ammonium, 447
PTR2 expression is downregulated via catabolite repression (50). Addition of leucine and 448
tryptophan promotes expression of PTR2. Thus, in the present study, Ptr2 proteins were 449
substantially expressed in SD medium that contained 90 µg•mL−1
leucine and 40 µg•mL−1
450
tryptophan, even though the medium contained rich nitrogen sources. Induction of PTR2 by 451
extracellular amino acids requires the SPS (Ssy1-Ptr3-Ssy5) sensor signal transduction system 452
(21). PTR2 transcription is also induced by dipeptides containing N-end rule amino acids (24, 453
25). The dipeptides bind to Ubr1 followed by degradation of the Cup9 repressor. Despite the 454
considerable amounts of transcriptional analyses, post-translational regulation of Ptr2 has yet 455
to be characterized. We showed that Ptr2 underwent rapid degradation when cells were 456
cultured in either SD or Ala–Trp medium, suggesting that the steady-state turnover rate of 457
Ptr2 is rapid but unaffected by external nitrogen species. Rapid degradation of Ptr2 depends 458
on Rsp5 ubiquitin ligase and its binding proteins Bul1 and Bul2 in a manner similar to amino 459
acid permeases Gap1 and Tat2, or uracil permease Fur4. This ubiquitination occurs most 460
probably at K16, K27, and K34 in the N-terminal cytoplasmic domain of Ptr2; however, 461
whether ubiquitin covalently binds to the lysines has not been tested. Ptr2 carrying the E92Q, 462
R93K, K205R, W362L, or E480D mutation was dysfunctional and eventually was degraded 463
through ubiquitination when cells were cultured in Ala–Trp medium. There are at least two 464
possibilities to account for the peptide-induced degradation of dysfunctional Ptr2. First, E92, 465
R93, K205, W362, and E480 are thought to reside in the substrate-binding pocket according 466
to the Ptr2 structural model. Therefore, the 5 mutations could abrogate Ptr2 to form a 467
substrate-binding occluded conformation, and hence, the 5 Ptr2 mutant proteins (binding 468
mutants, hereafter) might remain as the substrate-free outward-facing conformation. 469
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Eventually, Ptr2 in this conformation is regarded as a preferential target for Rsp5-dependent 470
ubiquitination. On the other hand, the remaining 9 Ptr2 mutant proteins, where most mutation 471
sites are located within the intracellular gate (gate mutants, hereafter), could form the 472
substrate-binding occluded conformation that may not be a preferential target for 473
ubiquitination. Second, the gate mutants retain marginal peptide importing activity because 474
addition of 100 µg•mL−1
Ala–Trp partially restored growth defects in ptr2 cells, which was 475
not the case in the binding mutants (Fig. 3B). In the gate mutant cells, internal tryptophan 476
might be sufficient to continue protein synthesis and prevent efficient ubiquitination of Ptr2. 477
In contrast, the binding mutant cells are depleted for tryptophan, and eventually downregulate 478
Ptr2 for efficient degradation. This is possible because degradation of the 5 binding mutants 479
was delayed in tryptophan prototrophic cells when moved to Ala–Trp medium (Fig. 4C). 480
Although these models do not explain why Ptr2 is stably expressed in peptide-free SD 481
medium, we speculate that a certain mechanism maintains Ptr2 expression insomuch as cell 482
growth is supported by free amino acids. 483
Addition of uracil induces rapid downregulation of the transporter Fur4 (51). The Fur4 484
structure was modeled by crystal structures of Mhp1, a bacterial homolog of Fur4. The 485
N-terminal domain of Mhp1, which is composed of 20 amino acid residues, runs parallel to 486
the membrane along a groove between the cytoplasmic loops. In the outward facing or ground 487
state of Mhp1, the N-terminal domain is kept in position by hydrogen-bond interactions with 488
the loops and the C-terminal domain; however, the interactions are attenuated in the 489
inward-facing state (51). A mechanism has been proposed that is based on a 490
conformation-sensing domain (loop interaction domain, LID) that regulates substrate-induced 491
downregulation and quality control in Fur4. The LID is located within the N-terminal 492
cytoplasmic tail that interacts with intermembrane loop regions, thereby stabilizing the 493
outward facing or ground state of Fur4. In the proposed model, conformational change to the 494
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inward-facing state caused by substrate binding or increased import activity in Fur4 transmits 495
to the LID, which becomes accessible to Rsp5 ubiquitin ligase (51). Although Ptr2 does not 496
contain the LID consensus motif, it might have a similar domain in the cytoplasmic tail that 497
senses the conformational status of this transporter. 498
Gap1 is the best-characterized amino acid permease for nitrogen-induced degradation. 499
When cells are grown on poor nitrogen sources, such as urea or proline, GAP1 transcription 500
increase and Gap1 accumulates in the plasma membrane. Upon addition of rich nitrogen 501
sources, such as ammonium ions or glutamate, Gap1 is ubiquitinated by Rsp5 on the 502
N-terminal K9 or K16, which is followed by delivery to the multivesiclular body and then to 503
the vacuole for degradation (40). As a new and distinct mechanism of amino acid dependent 504
downregulation of Gap1 activity, Gap1 itself is likely to be the sensor for high amino acid 505
levels. It was shown that an exogenously added rich mixture of amino acids (0.25% Casamino 506
acids) inactivated amino acid import through Gap1K9R-K16R
that resides at the plasma 507
membrane (52). A mutant Gap1A297V
imports glycine as efficiently as wild-type Gap1 does, 508
but it is unable to import basic amino acids such as citrulline, lysine, or arginine. Based on a 509
competitive import assay, basic amino acids appeared to bind to the active site of Gap1A297V
510
with the same affinity as the wild-type Gap1, but after substrate binding, Gap1A297V
cannot 511
undergo conformational changes necessary to complete the transport cycle (53). Although 512
Casamino acids inactivate glycine import through Gap1K9R-K16R-A297V
, basic amino acids have 513
no effect on glycine import through the mutant protein (53). Therefore, the amino 514
acid-dependent inactivation of Gap1 at the plasma membrane requires active transport of 515
amino acids through the permease. To validate the concept in Ptr2, we will examine whether 516
exogenously added peptides bind to the Ptr2 gate mutants but not to the binding mutants, and 517
whether the presumed conformational changes upon peptide binding could prevent Ptr2 518
ubiquitination. Peptides other than Ala–Trp and Ala–Ala that can be imported through the Ptr2 519
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binding mutants may not induce ubiquitin-dependent degradation of the transporter proteins. 520
521
ACKNOWLEDGEMENTS 522
We thank Masahiro Sugiyama and Mayu Miyachi for technical assistances; Keisuke Ito for 523
valuable discussions. This work was supported by grants from the Japan Society for the 524
Promotion of Science (No. 24580122 to F. Abe) and the Program for the Strategic Research 525
Foundation at Private Universities by the Ministry of Education, Culture, Sports, Science, and 526
Technology (No. 2013–2017). 527
528
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51. Keener JM, Babst M. 2013. Quality control and substrate-dependent downregulation of 670
the nutrient transporter Fur4. Traffic 14: 412–427. 671
52. Risinger AL, Cain NE, Chen EJ, Kaiser CA. 2006. Activity-dependent reversible 672
inactivation of the general amino acid permease. Mol. Biol. Cell 17: 4411–4119. 673
53. Cain NE, Kaiser CA. 2011. Transport activity-dependent intracellular sorting of the yeast 674
general amino acid permease. Mol. Biol. Cell 22: 1919–1929. 675
54. Simossis VA, Heringa J. 2005. PRALINE: A multiple sequence alignment toolbox that 676
integrates homology-extended and secondary structure information. Nucleic Acids Res. 677
33: W289–294. 678
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Table 1. Plasmids used in this study
Plasmid Description Source or Reference
pJJ280 TRP1 30
YCplac33 URA3 CEN4 31
pUA35 pTDH3-3HA-tTDH3, URA3 CEN4 This study
pKK1 GFP URA3 CEN4 This study
pPTR2-3HA PTR2-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-E92Q-3HA PTR2-E92Q-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-R93K-3HA PTR2-R93K-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-Y96A-3HA PTR2-Y96A-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-Y138A-3HA PTR2-Y138A-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-D149E-3HA PTR2-D149E-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-G168A-3HA PTR2-G168A-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-L196A-3HA PTR2-L196A-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-K205R-3HA PTR2-K205R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-S209A-3HA PTR2-S209A-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-F244L-3HA PTR2-F244L-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-F247L-3HA PTR2-F247L-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-W362L-3HA PTR2-W362L-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-E480D-3HA PTR2-E480D-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-G487A-3HA PTR2-G487A-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-L488A-3HA PTR2-L488A-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-M504L-3HA PTR2-M504L-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-F507W-3HA PTR2-F507W-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-T510V-3HA PTR2-T510V-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-G514A-3HA PTR2-G514A-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-K16R-3HA PTR2-K16R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-K27R-3HA PTR2-K27R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-K34R-3HA PTR2-K34R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-K77R-3HA PTR2-K77R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-3K>R-3HA PTR2-K16,27,34R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-4K>R-3HA PTR2-K16,27,34,77R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-E92Q-3K>R-
3HA PTR2-E92Q-K16,27,34R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-R93K-3K>R-
3HA PTR2-R93K-K16,27,34R-3HA driven by PTR2 promoter, URA3 CEN4 This study
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pPTR2-K205R-3K>R
-3HA PTR2-K205R-K16,27,34R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-W362L-3K>R
-3HA PTR2-W362L-K16,27,34R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-E480D-3K>R
-3HA PTR2-E480D-K16,27,34R-3HA driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-GFP PTR2-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-E92Q-GFP PTR2-E92Q-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-R93K-GFP PTR2-R93K-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-Y96A-GFP PTR2-Y96A-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-Y138A-GFP PTR2-Y138A-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-D149E-GFP PTR2-D149E-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-G168A-GFP PTR2-G168A-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-L196A-GFP PTR2-L196A-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-K205R-GFP PTR2-K205R-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-S209A-GFP PTR2-S209A-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-F244L-GFP PTR2-F244L-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-F247L-GFP PTR2-F247L-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-W362L-GFP PTR2-W362L-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-E480D-GFP PTR2-E480D-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-G487A-GFP PTR2-G487A-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-L488A-GFP PTR2-L488A-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-M504L-GFP PTR2-M504L-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-F507W-GFP PTR2-F507W-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-T510V-GFP PTR2-T510V-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-G514A-GFP PTR2-G514A-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-3K>R-GFP PTR2-K16,27,34R-GFP driven by PTR2 promoter, URA3 CEN4 This study
pPTR2-4K>R-GFP PTR2-K16,27,34,77R-GFP driven by PTR2 promoter, URA3 CEN4 This study
679
680
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Table 2. Mutated amino acid residues in Ptr2 and corresponding PepTst residues
Ptr2
mutation TMD1)
Ptr2
function2)
Corresponding PepTst
residue Description
None
++
E92Q 1 – E25 The ExxERFxYY motif
R93K 1 – R26 The ExxERFxYY motif
Y96A 1 + Y29 The ExxERFxYY motif
Y138A 2 +/– Y68 Recognition of C-terminus of substrates
D149E 2 ++ D79 Highly conserved residue
G168A 3 ++ G98 Highly conserved residue
L196A 4 ++ L117 Highly conserved residue
K205R 4 – K124 Interaction with the ExxERFxYY motif
S209A 4 ++ S130 Intracellular gate
F244L 5 – F148 Highly conserved residue
F247L 5 +/– F151 Intracellular gate
W362L 7 – W296 Highly conserved residue
E480D 10 – E400 Recognition of N-terminus of substrates
G487A 10 +/– G407 Intracellular gate
L488A 10 +/– L408 Intracellular gate
M504L 11 – M424 Intracellular gate
F507W 11 – W427 Intracellular gate
T510V 11 – S430 Interaction with W362
G514A 11 ++ G434 Highly conserved residue
1) TMDs were assigned based on the crystallographic structure of PepTst
(PDB: 4APS).
2) Ptr2 function was evaluated by the complementation test for growth of ptr2 cells in Ala-Trp medium.
–, no growth or impaired growth in 20 g•mL–1 Ala-Trp medium (< 10% of the OD600 value with respect to the wild-type cells)
+/–, impaired growth in 20 g•mL–1 Ala-Trp medium (10–50% of the OD600 value with respect to the wild-type cells) or growth defect is
partially rescued in 100 g•mL–1 Ala-Trp medium
+, moderately impaired growth in 20 g•mL–1 Ala-Trp medium (50–70% of the OD600 value with respect to the wild-type cells)
++, growth as the wild-type cells in 20 g•mL–1 Ala-Trp medium (> 70% of the OD600 value with respect to the wild-type cells)
681
682
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FIGURE LEGENDS 683
684
Figure 1. Multiple sequence alignments of TMDs for some peptide transporters. The 685
alignments were constructed for ScPtr2 (S. cerevisiae), CaPtr2 (C. albicans) hPepT1, and 686
hPepT2 (H. sapiens), PepTso (S. oneidensis), PepTst (S. thermophilus), and GkPOT (G. 687
kaustophilus) using the PRALINE multiple sequence alignment program (54). TMD -helices 688
were assigned based on the crystallographic structure of PepTst (PDB: 4APS) (28). Amino 689
acid residues that mutated in this study are marked with an asterisk (*) above the residue, and 690
those that reside in the extracellular gate in bacterial peptide transporters are marked with a 691
dot (•). 692
693
Figure 2. A structural model of Ptr2. The structure of Ptr2 was based on the crystal structure 694
of PepTst inward-open conformation (PDB: 4APS) as a template (28). Amino acid residues 695
that were mutated in this study are indicated. 696
697
Figure 3. Effects of Ptr2 mutations on cell growth. 3HA-tagged wild-type or Ptr2 mutant 698
proteins were expressed in ptr2Δ cells. The cells were incubated at 25 °C for 20 h in (A) SD 699
(40 µg•mL−1
tryptophan), (B) Ala–Trp medium containing 20 or 100 µg•mL−1
Ala–Trp in the 700
absence of free tryptophan, or (C) Leu–Trp medium containing 23 µg•mL−1
Leu–Trp in the 701
absence of free tryptophan. The starting OD600 value was 0.04. 702
703
Figure 4. Expression of Ptr2 mutant proteins. 3HA-tagged wild-type or Ptr2 mutant proteins 704
were expressed in (A) ptr2Δ trp1 cells, (B) PTR2 trp1 cells, or (C) ptr2Δ TRP1 cells. 705
Exponentially growing cells in SD medium were moved to Ala–Trp (AW) medium (20 706
µg•mL−1
) and incubated for an additional 5 h. Whole cell extracts were subjected to Western 707
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blot analysis to detect Ptr2-3HA and Adh1 using specific antibodies. Adh1 was used as a 708
loading control. 709
710
Figure 5. Localization of Ptr2 mutant proteins in ptr2Δ cells. (A) GFP-tagged wild-type or 711
Ptr2 mutant proteins were expressed in ptr2Δ cells. Exponentially growing cells in SD 712
medium were moved to Ala–Trp (AW) medium (20 µg•mL−1
) and incubated for an additional 713
5 h. The cells were imaged in a fluorescence microscope. Ptr2 in the plasma membrane are 714
shown in arrowheads. Ptr2R93K
-GFP and Ptr2W362L
-GFP were predominantly localized in the 715
vacuole, although they remained in the plasma membrane in some of the cells (~25%, lower 716
panels in R93K and W362L). (B) Whole cell extracts were subjected to Western blot analysis 717
to detect Ptr2-GFP and Adh1 using specific antibodies. Adh1 was used as a loading control. 718
719
Figure 6. Effects of Ptr2 mutations on Ala–Ala uptake. 3HA-tagged wild-type or Ptr2 mutant 720
proteins were expressed in ptr2Δ cells. (A) Competition import assay for [3H-] labeled Ala–721
Ala with nonlabeled Ala–Ala or Ala–Trp. The wild-type cells incubated for 10 min with [3H-] 722
labeled Ala–Ala in the presence of 72.6 M nonlabeled Ala–Ala (1×), or additional 723
nonlabeled Ala–Ala or Ala–Trp. (B) Effects of Ptr2 mutations on Ala–Ala uptake. Cells were 724
incubated in [3H-] labeled Ala–Ala in the presence of 72.6 M nonlabeled Ala–Ala. Data are 725
expressed as mean values of amino acid incorporated (pmol•107
cells−1
min−1
) with standard 726
deviations obtained from three independent experiments. Inset, time dependence of Ala-Ala 727
uptake in the wild-type cells. 728
729
Figure 7. Ubiquitin-dependent degradation of Ptr2 proteins. (A) Rapid degradation of Ptr2 in 730
the wild-type cells. (B) Stabilization of Ptr2 in the HPG1-1 (Rsp5P514T
) and the bul1Δbul2Δ 731
mutants cultured in SD medium. Whole cell extracts were prepared after adding 732
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cycloheximide. Western blot analysis was performed to detect Ptr2-3HA and Adh1 using 733
specific antibodies. Adh1 was used as a loading control. 734
735
Figure 8. Determination of lysine residues required for ubiquitin-dependent Ptr2 degradation. 736
(A) Whole cell extracts were prepared from ptr2Δ cells expressing Ptr2-3HA with single (top) 737
or multiple (bottom) K > R substitutions. SD medium was used for culture. Western blot 738
analysis was performed to detect Ptr2-3HA and Adh1 using specific antibodies. Adh1 was 739
used as a loading control. (B) The OD600 values were measured after individual strains were 740
grown in Ala–Trp medium (20 µg•mL−1
) at 25 °C for 20 h. The starting OD600 value was 0.04. 741
(C) GFP-tagged Ptr2 proteins with multiple K > R substitutions were expressed in ptr2Δ cells. 742
Exponentially growing cells in SD medium were moved to Ala–Trp medium (20 µg•mL−1
) 743
and incubated for an additional 5 h. The cells were imaged using a fluorescence microscope. 744
745
Figure 9. Ubiquitin-dependent degradation of dysfunctional Ptr2 proteins. (A) 3HA-tagged 746
wild-type or Ptr2 mutant proteins were expressed in ptr2Δ cells. Cycloheximide was added to 747
the cells in SD medium for 2 h. 3HA-tagged wild-type or Ptr2 mutant proteins were expressed 748
in (B) PTR2 or PTR2 HPG1-1 cells and (C) PTR2 or ptr2Δ HPG1-1 cells. (D) 3HA-tagged 749
wild-type or Ptr2 mutant proteins combined with the 3K > R substitution were expressed in 750
ptr2 cells. Exponentially growing cells in SD medium were moved to Ala–Trp (AW) 751
medium (20 µg•mL−1
) and incubated for an additional 5 h. Whole cell extracts were subjected 752
to Western blot analysis to detect Ptr2-3HA and Adh1 using specific antibodies. Adh1 was 753
used as a loading control. 754
755
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TMD1 TMD5
ScPtr2CaPtr2hPepT1hPepT2PepTsoPepTstGkPOT
7893
114129
1242101121
4373464757
ScPtr2CaPtr2hPepT1hPepT2PepTsoPepTstGkPOT
236251
269282
155176142140150
187208174172182
** * ● * *
ScPtr2CaPtr2hPepT1hPepT2PepTsoPepTst
123138
151166
49795353
771078181
ScPtr2CaPtr2hPepT1hPepT2PepTsoPepTst
350365
378393
282301300284
310329328312
* * * ●TMD2 TMD7
GkPOT 63 91
ScPtr2CaPtr2hPepT1hPepT2
154169
174189
80110
100130
pGkPOT 294 322
ScPtr2CaPtr2hPepT1hPepT2
466481
498513
581608
613640
* * **TMD3 TMD10
PepTsoPepTstGkPOT
848494
104104114
ScPtr2C Pt 2
187 215
PepTsoPepTstGkPOT
405386399
437418431
ScPtr2 503 527* * * * * * *
TMD4 TMD11
CaPtr2hPepT1hPepT2PepTsoPepTstGkPOT
202 230122143109108118
150171137136146
CaPtr2hPepT1hPepT2PepTsoPepTstGkPOT
518 542618645442423436
642669479447460
Unconserved Conserved
Fig. 1. Kawai et al.
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Top Side
M504T510G514 Y138 T510 G514
F244
Top Side
F507
W362D149
F247
G168
362L488
G487
Y96L196
G168
L196Y96Y138E480
W362
G487
E480 E92
R93 E92
R93F507
K205S209 F247
F244D149
K205S209M504L488
G487
Putative trajectoryTMD 1 2 3 4 5 6 7 8 9 10 11 12
Fig. 2. Kawai et al.
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3 3 320 μg•mL–1
100 μg•mL–1
Ala-TrpA B
23 μg•mL–1
Leu-TrpSDC
2 2 2
D60
0at
20
h
D60
0at
20
h
D60
0at
20
h
100 μg mL
0
1
0
1
WT or 2Q 3K 6A 8A 9E 8A 6A 5R 9A 4L 7L 2L 0D 7A 8A 4L W 0V 4A
0
1
WT or Q 3K 6A 8A 9E 8A 6A 5R 9A 4L 7L 2L 0D 7A 8A 4L W 0V 4A
OD
OD
OD
Wve
cto
E92
R93 Y9
6Y1
38D
149
G16
8L1
96K
205
S209
F244
F247
W36
2E4
80G
487
L488
M50
4F5
07W
T510
G51
4 Wve
cto
E92
R9 3 Y9
6Y1
38D
149
G16
8L1
96K
205
S209
F244
F247
W36
2E4
80G
487
L488
M50
4F5
07W
T510
G51
4
Fig. 3. Kawai et al.
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SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW
Adh1
Ptr2-3HA
Adh1
SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD
AW
SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW
Adh1
Adh1
SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD
AW
A
ptr
2D
trp
1 c
ells
B
PT
R2
trp
1 c
ells
Fig. 4. Kawai et al.
Ptr2-3HA
Ptr2-3HA
Ptr2-3HA
ptr2D trp1 cells ptr2D TRP1 cells
SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD
AW
Adh1
Ptr2-3HA
C
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GFP DIC
AW
Ptr2-GFP
Ptr2-GFP
SD AW
WT
E92Q
R93K
Y96A
Y138A
D149E
G168
A
L196A
K205R
S209A
F244L
F247L
W362L
E480D
G487A
L488A
M504L
F507W
T510V
G514A
SD AW SD AW SD AW SD AW SD AW SD AW SD AW
SD AW SD AW SD AW SD AW SD AW SD AW SD AW
SD AW SD AW SD AW SD AW SD AW SD AW SD AW
GFP DIC GFP DIC GFP DIC
SD SD AW
Ptr2-GFP
75kDa
63kDa
75kDa
63kDa
75kDa
63kDa
Adh1
Adh1
Adh1
ptr2D cells ptr2D cells
Partial
digests
Partial
digests
Partial
digests
Fig. 5. Kawai et al.
A B
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A B
50
60
70ce
lls–1
min
–1)
4000
5000
a(D
PM)
Ala-Ala
Ala-Trp300
400
mol
107
cells
–1)
30
40
50
Ala
(pm
ol10
7c
2000
3000
of [3
H] A
la-A
la
0
100
200
5 5 15Ala
-Ala
upta
ke (p
m
0
10
20
Upt
ake
of A
la-A
1000Upt
ake
o -5 5 15Time (min)
0U01 × 1.5 × 2 × 3×Non-labeled Ala-Ala or Ala-Trp
ptr2Δ cells
Fig. 6. Kawai et al.
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Adh1
Ptr2-3HA
Adh1
Ptr2-3HA
0 1 2 0 1 2
SD Ala-Trp
CHX (h)
0 1 2 0 1 2 0 1 2 CHX (h)
RSP5 (WT) HPG1-1 bul1Dbul2D
A
B
PTR2 (WT) cells
Fig. 7. Kawai et al.
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0 2 0 2 0 2 0 2 0 2 WT K16R K27R K34R K77R
CHX (h)
Ptr2-3HA
A
WT K34R K16, 34R K27, 34R 3K>R 4K>R
Ptr2 3HA
Adh1
cells
0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 , ,
CHX (h)
Ptr2-3HA
ptr2
Δ
Adh1
B C
2
3
20 h
2
3
20
B ptr2Δ cells ptr2Δ cellsGFP DIC
SDGFP DIC
Ala–Trp
3K>R
C
1
OD
600
at 2
1
OD
600
at 2
4K>R
0 0
Fig. 8. Kawai et al.
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PTR2 cells
WT WT E92Q R93K K205R W362L E480DWT E92Q R93K K205R W362L E480D
PTR2 HPG1-1 cellsA ptr2Δ cells BWT WT E92Q R93K K205R W362L E480DWT E92Q R93K K205R W362L E480D
0 2 0 2 0 2 0 2 0 2 0 2
Ptr2-3HA
Adh1
CHX (h) SD AW SD AW SD AW SD AW SD AW SD AW SD AW
Ptr2-3HA
Adh1
ptr2Δ HPG1-1 cellsPTR2 cellsC
+ 3K>Rptr2Δ cellsD
SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AW SD AWWT WT E92Q R93K K205R W362L E480D
Ptr2-3HA
Adh1
Ptr2-3HA
Adh1
WT WT E92Q R93K K205R W362L E480D
Adh1
Fig. 9. Kawai et al.
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