The sucrose permease of Escherichia coli: functional significance of cysteine residues and properties of a cysteine-less transporter.
The sucrose (CscB) permease belongs to the oligosaccharide:H(+) symporter family of the Major Facilitator Superfamily and is homologous to the lactose permease from Escherichia coli. Sucrose transport in cells expressing sucrose permease is completely inhibited by N-ethylmaleimide (NEM), suggesting that one or more of the seven native Cys residues may be important for transport. In this paper, each Cys residue was individually replaced with Ser, and transport activity, membrane expression, and NEM sensitivity are documented. All seven single Cys-->Ser mutants are expressed normally in the membrane and catalyze sucrose transport with activities ranging from 80% to 180% of wild type. Six of the seven Ser mutants are completely inactivated by NEM, while Cys122-->Ser permease is insensitive to the sulfhydryl reagent, indicating that NEM inhibition results from alkylation of Cys122. Subsequently, a sucrose permease devoid of Cys residues (Cys-less) was constructed in which all Cys residues were replaced with Ser simultaneously by using a series of overlap-extension PCRs. Membrane expression and kinetic parameters for Cys-less [K(m) 4.8 mM, V(max) 192 nmol min(-1) (mg of protein)(-1)] are essentially identical to those of wild type [K(m) 5.4 mM, V(max) 196 nmol min(-1) (mg of protein)(-1)]. However, Cys-less permease catalyzes sucrose accumulation to steady-state levels that are approximately 2-fold higher than those of wild type. As anticipated, Cys-less permease is completely resistant to NEM inhibition. The observations demonstrate that Cys residues play no functional role in sucrose permease. Furthermore, the approach described to create the Cys-less transporter is generally applicable to other proteins. An application of Cys-less permease in the study of the substrate binding site is presented in the accompanying paper.
Sahin-Tóth
M
M
Howard Hughes Medical Institute, Departments of Physiology and Microbiology & Molecular Genetics, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Frillingos
S
S
Lawrence
M C
MC
Kaback
H R
HR
D000478:Alkylation; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000235:genetics; D013395:Sucrose / Q000037:antagonists & inhibitors / Q000378:metabolism
D029968:Escherichia coli Proteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D012694:Serine; C056379:cscB protein, E coli; D013395:Sucrose; D003545:Cysteine; D005033:Ethylmaleimide