Amino acid . | Mutation . | Effect of mutation . | Proposed function of the residue . | Experimental system . | Reference . |
---|---|---|---|---|---|
Human PepT1 | |||||
Y12 | Y12A | Mutation has a modest effect on Gly-Sar uptake and primarily affects the Vmax value of the translocation process | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998) | |
H57 | H57Q | Mutants have no detectable peptide transport activity | H57 is involved in binding and translocation of H+ cotransported with the peptide and is a principal proton-binding site | Expression in Xenopus oocytes and electrophysiology; transfection of HeLa cells and radioactive peptide uptake; transfection of HEK293 cells and radioactive peptide uptake | (Fei et al., 1997; Uchiyama et al., 2003) |
H57N | |||||
H57A | Mutants have no detectable peptide transport activity; moreover, mutant H57R evokes increasing steady-state currents with gradual increase of the pH(5.0–8.5) | ||||
H57R | |||||
H57K | |||||
H121 | H121A | Mutants show reduced uptake of Gly-Sar by 43% (H121A), 45% (H121R), 75%(H121K); moreover, mutants H121R and H121K show a significant decrease of Gly–Sar uptake at pH 7.4 and 8.5 compared to pH 6.0 | H121 is probably involved in substrate recognition and is not involved in H+ binding | Expression in Xenopus oocytes and electrophysiology; transfection of HEK293 cells and radioactive peptide uptake | (Uchiyama et al., 2003) |
H121R | |||||
H121K | |||||
S164 | S164C | Mutant is not expressed on the plasma membrane | This amino acid position is responsible for incorrect packaging and/or transport of the protein to the plasma membrane | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003a) |
Y167 | Y167A | Uptake of Gly–Sar is abolished | This residue has an essential role in dipeptide uptake (due to the unique chemistry of its phenolic side chain) | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998; Kulkarni et al., 2003a; Yeung et al., 1998) |
Y167F | |||||
Y167H | |||||
Y167S | |||||
Y167C | |||||
L168 | L168C | Mutant is not expressed on the plasma membrane | This amino acid position is responsible for incorrect packaging and/or transport of the protein to the plasma membrane | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003a) |
N171 | N171C | Uptake of Gly–Sar is abolished | This amino acid plays a critical role in substrate binding | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003a) |
G173 | G173C | Mutant is not expressed on the plasma membrane | This amino acid position is responsible for incorrect packaging and/or transport of the protein to the plasma membrane | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003a) |
Human PepT1 | |||||
S174 | S174C | Uptake of Gly–Sar is abolished | This amino acid plays a critical role in substrate binding | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003a) |
I179 | I179C | Mutant is not expressed on the plasma membrane | This amino acid position is responsible for incorrect packaging and/or transport of the protein to the plasma membrane | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003a) |
P182 | P182C | Mutant shows ∼40% of Gly-Sar uptake | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003a) | |
R282 | R282A | Mutations have a modest effect on Gly–Sar uptake | The positive charge is important at amino acid position 282. A salt-bridge between R282–D341 may play a role in maximizing the efficiency of substrate translocation. | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998, Kulkarni et al., 2007; Kulkarni et al., 2003b) |
R282C | |||||
R282K | |||||
R282E | Mutants show significantly reduced uptake of Gly–Sar | ||||
R282D | |||||
Y287 | Y287C | Mutant is not expressed on the plasma membrane | Single cysteine mutation at this position is responsible for incorrect synthesis and/or misfolding of the mutated protein | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003b) |
M292 | M292C | Mutant is not expressed on the plasma membrane | Single cysteine mutation at this position is responsible for incorrect synthesis and/or misfolding of the mutated protein | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003b) |
F293 | F293C | Mutant displays negligible uptake of Gly–Sar | This residue probably plays a structural role in the transporter function | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003b) |
W294 | W294A | Mutant shows reduced uptake of Gly–Sar (∼8%); in particular,mutation has a significant effect on the Michaelis–Menten Km value | This residue plays a role in maintaining the structural integrity of the protein. The larger size of the cysteine side chain, compared with that of alanine, sufficiently reflects the steric bulk of the tryptophan side chain and better maintains the correct helical packing | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998; Kulkarni et al., 2003b) |
W294C | Mutant does not show reduced uptake of Gly–Sar | ||||
L296 | L296C | Mutant displays negligible uptake of Gly–Sar | This residue probably plays a structural role in the transporter function | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003b) |
F297 | F297C | Mutant displays negligible uptake of Gly–Sar | This residue probably plays a structural role in the transporter function | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003b) |
D341 | D341A | Mutations do not show significantly reduced uptake of Gly–Sar | The negative charge is important at amino acid position 341. A salt-bridge between R282–D341 may play a role in maximizing the efficiency of substrate translocation | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2007) |
D341E | |||||
D341K | Mutants show significantly reduced uptake of Gly–Sar | ||||
D341R | |||||
Human PepT1 | |||||
P586 | P586L | Mutant shows reduced transport capacity, lower protein level and lower plasma membrane expression | P586 may have profound effects on translation, degradation, and/or membrane insertion | Transfection of HeLa cells, radioactive peptide uptake and immunocytochemical and Western blot analyses | (Zhang et al., 2004b) |
E595 | E595A | Mutant shows reduced uptake of Gly–Sar (∼95%) | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998) | |
Rabbit PepT1 | |||||
Y56 | Y56A | Mutant Y56F exhibits slightly decreased functional activities, while Y56A exhibits no significant activities | This aromatic residue stabilizes the charge on H+ when interacting with H57 | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Chen et al., 2000) |
Y56F | |||||
H57 | H57R | Mutant has no detectable peptide transport activity | H57 residue is involved in the binding of H+ to the transporter | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Chen et al., 2000) |
Y64 | Y64A | Mutant Y64F exhibits slightly decreased functional activities, while Y64A exhibits no significant activities | This aromatic residue stabilizes the charge on H+ when interacting with H57 | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Chen et al., 2000) |
Y64F | |||||
H121 | H121R | Affinity of H121 mutants for peptide substrates decreases depending on the charge of the substrate | H121 is involved in substrate recognition | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Chen et al., 2000) |
H121C | |||||
R282 | R282E | Mutation uncouples the H+-peptide cotransport and creates a peptide-gated cation channel in the protein | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Meredith, 2004) | |
W294 | W294F | Mutant does not show any peptide uptake and does not produce any depolarization of membrane potential | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Panitsas et al., 2006) | |
Rat PepT1 | |||||
H57 | H57Q | Uptake of Gly–Sar is abolished | H57 is involved in substrate binding and/or is responsible for intrinsic activity of the transporter | Expression in Xenopus oocytes and radioactive peptide uptake | (Terada et al., 1996) |
H121 | H121Q | Uptake of Gly–Sar is abolished | H121 is involved in substrate binding and/or is responsible for intrinsic activity of the transporter | Expression in Xenopus oocytes and radioactive peptide uptake | (Terada et al., 1996) |
Amino acid . | Mutation . | Effect of mutation . | Proposed function of the residue . | Experimental system . | Reference . |
---|---|---|---|---|---|
Human PepT1 | |||||
Y12 | Y12A | Mutation has a modest effect on Gly-Sar uptake and primarily affects the Vmax value of the translocation process | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998) | |
H57 | H57Q | Mutants have no detectable peptide transport activity | H57 is involved in binding and translocation of H+ cotransported with the peptide and is a principal proton-binding site | Expression in Xenopus oocytes and electrophysiology; transfection of HeLa cells and radioactive peptide uptake; transfection of HEK293 cells and radioactive peptide uptake | (Fei et al., 1997; Uchiyama et al., 2003) |
H57N | |||||
H57A | Mutants have no detectable peptide transport activity; moreover, mutant H57R evokes increasing steady-state currents with gradual increase of the pH(5.0–8.5) | ||||
H57R | |||||
H57K | |||||
H121 | H121A | Mutants show reduced uptake of Gly-Sar by 43% (H121A), 45% (H121R), 75%(H121K); moreover, mutants H121R and H121K show a significant decrease of Gly–Sar uptake at pH 7.4 and 8.5 compared to pH 6.0 | H121 is probably involved in substrate recognition and is not involved in H+ binding | Expression in Xenopus oocytes and electrophysiology; transfection of HEK293 cells and radioactive peptide uptake | (Uchiyama et al., 2003) |
H121R | |||||
H121K | |||||
S164 | S164C | Mutant is not expressed on the plasma membrane | This amino acid position is responsible for incorrect packaging and/or transport of the protein to the plasma membrane | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003a) |
Y167 | Y167A | Uptake of Gly–Sar is abolished | This residue has an essential role in dipeptide uptake (due to the unique chemistry of its phenolic side chain) | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998; Kulkarni et al., 2003a; Yeung et al., 1998) |
Y167F | |||||
Y167H | |||||
Y167S | |||||
Y167C | |||||
L168 | L168C | Mutant is not expressed on the plasma membrane | This amino acid position is responsible for incorrect packaging and/or transport of the protein to the plasma membrane | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003a) |
N171 | N171C | Uptake of Gly–Sar is abolished | This amino acid plays a critical role in substrate binding | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003a) |
G173 | G173C | Mutant is not expressed on the plasma membrane | This amino acid position is responsible for incorrect packaging and/or transport of the protein to the plasma membrane | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003a) |
Human PepT1 | |||||
S174 | S174C | Uptake of Gly–Sar is abolished | This amino acid plays a critical role in substrate binding | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003a) |
I179 | I179C | Mutant is not expressed on the plasma membrane | This amino acid position is responsible for incorrect packaging and/or transport of the protein to the plasma membrane | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003a) |
P182 | P182C | Mutant shows ∼40% of Gly-Sar uptake | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003a) | |
R282 | R282A | Mutations have a modest effect on Gly–Sar uptake | The positive charge is important at amino acid position 282. A salt-bridge between R282–D341 may play a role in maximizing the efficiency of substrate translocation. | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998, Kulkarni et al., 2007; Kulkarni et al., 2003b) |
R282C | |||||
R282K | |||||
R282E | Mutants show significantly reduced uptake of Gly–Sar | ||||
R282D | |||||
Y287 | Y287C | Mutant is not expressed on the plasma membrane | Single cysteine mutation at this position is responsible for incorrect synthesis and/or misfolding of the mutated protein | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003b) |
M292 | M292C | Mutant is not expressed on the plasma membrane | Single cysteine mutation at this position is responsible for incorrect synthesis and/or misfolding of the mutated protein | Transfection of HEK293 cells and immunofluorescence experiments | (Kulkarni et al., 2003b) |
F293 | F293C | Mutant displays negligible uptake of Gly–Sar | This residue probably plays a structural role in the transporter function | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003b) |
W294 | W294A | Mutant shows reduced uptake of Gly–Sar (∼8%); in particular,mutation has a significant effect on the Michaelis–Menten Km value | This residue plays a role in maintaining the structural integrity of the protein. The larger size of the cysteine side chain, compared with that of alanine, sufficiently reflects the steric bulk of the tryptophan side chain and better maintains the correct helical packing | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998; Kulkarni et al., 2003b) |
W294C | Mutant does not show reduced uptake of Gly–Sar | ||||
L296 | L296C | Mutant displays negligible uptake of Gly–Sar | This residue probably plays a structural role in the transporter function | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003b) |
F297 | F297C | Mutant displays negligible uptake of Gly–Sar | This residue probably plays a structural role in the transporter function | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2003b) |
D341 | D341A | Mutations do not show significantly reduced uptake of Gly–Sar | The negative charge is important at amino acid position 341. A salt-bridge between R282–D341 may play a role in maximizing the efficiency of substrate translocation | Transfection of HEK293 cells and radioactive peptide uptake | (Kulkarni et al., 2007) |
D341E | |||||
D341K | Mutants show significantly reduced uptake of Gly–Sar | ||||
D341R | |||||
Human PepT1 | |||||
P586 | P586L | Mutant shows reduced transport capacity, lower protein level and lower plasma membrane expression | P586 may have profound effects on translation, degradation, and/or membrane insertion | Transfection of HeLa cells, radioactive peptide uptake and immunocytochemical and Western blot analyses | (Zhang et al., 2004b) |
E595 | E595A | Mutant shows reduced uptake of Gly–Sar (∼95%) | Transfection of HEK293 cells and radioactive peptide uptake | (Bolger et al., 1998) | |
Rabbit PepT1 | |||||
Y56 | Y56A | Mutant Y56F exhibits slightly decreased functional activities, while Y56A exhibits no significant activities | This aromatic residue stabilizes the charge on H+ when interacting with H57 | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Chen et al., 2000) |
Y56F | |||||
H57 | H57R | Mutant has no detectable peptide transport activity | H57 residue is involved in the binding of H+ to the transporter | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Chen et al., 2000) |
Y64 | Y64A | Mutant Y64F exhibits slightly decreased functional activities, while Y64A exhibits no significant activities | This aromatic residue stabilizes the charge on H+ when interacting with H57 | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Chen et al., 2000) |
Y64F | |||||
H121 | H121R | Affinity of H121 mutants for peptide substrates decreases depending on the charge of the substrate | H121 is involved in substrate recognition | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Chen et al., 2000) |
H121C | |||||
R282 | R282E | Mutation uncouples the H+-peptide cotransport and creates a peptide-gated cation channel in the protein | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Meredith, 2004) | |
W294 | W294F | Mutant does not show any peptide uptake and does not produce any depolarization of membrane potential | Expression in Xenopus oocytes and radioactive peptide uptake or electrophysiology | (Panitsas et al., 2006) | |
Rat PepT1 | |||||
H57 | H57Q | Uptake of Gly–Sar is abolished | H57 is involved in substrate binding and/or is responsible for intrinsic activity of the transporter | Expression in Xenopus oocytes and radioactive peptide uptake | (Terada et al., 1996) |
H121 | H121Q | Uptake of Gly–Sar is abolished | H121 is involved in substrate binding and/or is responsible for intrinsic activity of the transporter | Expression in Xenopus oocytes and radioactive peptide uptake | (Terada et al., 1996) |
Amino acid notation follows the single letter code