Cysteine 144 in the Third Transmembrane Domain of the Creatine Transporter Is Located Close to a Substrate-binding Site

All creatine transporters contain a cysteine residue (Cys144) in the third transmembrane domain that is not present in other members of the Na+,Cl−-dependent family of neurotransmitter transporters. Site-directed mutagenesis and reaction with methane thiosulfonates were used to investigate the importance of Cys144for transporter function. Replacement of Cys144with Ser did not significantly affect the kinetics or activity of the transporter, whereas a C144A mutant had a higherKm(0.33 compared with 0.18 mm). Substitution of Cys144with Leu gave a mutant with a 5-fold higherKmand a reduced specificity for substrate. Low concentrations of 2-aminoethyl methanethiosulfonate (MTSEA) resulted in rapid inactivation of the creatine transporter. The C144S mutant was resistant to inactivation, indicating that modification of Cys144was responsible for the loss of transport activity. Creatine and analogues that function as substrates of the creatine transporter were able to protect from MTSEA inactivation. Na+and Cl−ions were not necessary for MTSEA inactivation, but Na+was found to be important for creatine protection from inactivation. Our results indicate that cysteine 144 is close to the binding site or part of a permeation channel for creatine.

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Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1

Biochemical Journal ◽

10.1042/bj3440205 ◽

1999 ◽

Vol 344 (1) ◽

pp. 205-209 ◽

Cited By ~ 9

Author(s):

Ana M. PAJOR ◽

Sally J. KRAJEWSKI ◽

Nina SUN ◽

Rama GANGULA

Keyword(s):

Protein Trafficking ◽

Direct Relationship ◽

Transmembrane Domain ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Transport Activity ◽

Specific Reagent

The role of cysteine residues in the Na+/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.

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LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor

Applied and Environmental Microbiology ◽

10.1128/aem.01293-16 ◽

2016 ◽

Vol 82 (17) ◽

pp. 5364-5374 ◽

Cited By ~ 8

Author(s):

Marija Miljkovic ◽

Gordana Uzelac ◽

Nemanja Mirkovic ◽

Giulia Devescovi ◽

Dzung B. Diep ◽

...

Keyword(s):

Amino Acids ◽

Transmembrane Domain ◽

Transmembrane Helix ◽

Sugar Transport ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Bacteriocin Activity ◽

Binding Studies ◽

Content Type ◽

The Third

ABSTRACTThe Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. AlthoughyvjBis highly conserved in the genusLactococcus, the bacteriocin appears to be active only against the subspeciesL. lactissubsp.lactis. Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp25) and terminal alanine (Ala30). It was also shown that the distance between Trp25and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr356) and alanine (Ala353).In vitroexperiments showed that LsbB could interact with both YvjBMNand YvjBMG, but the strength of interaction is significantly less with YvjBMG.In vivoexperiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN.IMPORTANCEThe antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr356and Ala353residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

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Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

Applied and Environmental Microbiology ◽

10.1128/aem.02197-14 ◽

2014 ◽

Vol 80 (20) ◽

pp. 6549-6559 ◽

Cited By ~ 6

Author(s):

Sabrina Wemhoff ◽

Roland Klassen ◽

Friedhelm Meinhardt

Keyword(s):

Kluyveromyces Lactis ◽

Killer Toxin ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Target Cells ◽

Directed Mutagenesis ◽

Content Type ◽

Protein Toxin ◽

Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

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Protein-ligand interactions in the lysine-binding site of plasminogen kringle 4 are different in crystal and solution. Electrostatic interactions studied by site-directed mutagenesis exclude Lys35 as an important acceptor in solution

Biochemistry ◽

10.1021/bi00211a010 ◽

1993 ◽

Vol 32 (48) ◽

pp. 13019-13025 ◽

Cited By ~ 6

Author(s):

Peter Reinholt Nielsen ◽

Katja Einer-Jensen ◽

Thor L. Holtet ◽

Bente Damm Andersen ◽

Flemming M. Poulsen ◽

...

Keyword(s):

Binding Site ◽

Electrostatic Interactions ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Protein Ligand Interactions ◽

Ligand Interactions

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Ethanol Inhibition ofN-Methyl-d-aspartate Receptors Is Reduced by Site-directed Mutagenesis of a Transmembrane Domain Phenylalanine Residue

Journal of Biological Chemistry ◽

10.1074/jbc.m102800200 ◽

2001 ◽

Vol 276 (48) ◽

pp. 44729-44735 ◽

Cited By ~ 91

Author(s):

Kimberly M. Ronald ◽

Tooraj Mirshahi ◽

John J. Woodward

Keyword(s):

Transmembrane Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Ethanol Inhibition ◽

Phenylalanine Residue

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Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

Biochemical Journal ◽

10.1042/bj3020355 ◽

1994 ◽

Vol 302 (2) ◽

pp. 355-361 ◽

Cited By ~ 31

Author(s):

K Inukai ◽

T Asano ◽

H Katagiri ◽

M Anai ◽

M Funaki ◽

...

Keyword(s):

Glucose Transporter ◽

Cytochalasin B ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Site Directed Mutagenesis ◽

Transport Activity ◽

Wild Type ◽

In The Wild ◽

Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

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