ABCMdb

PMID: 12421811

Abdel-Dayem M, Basquin C, Pourcher T, Cordat E, Leblanc G
Cytoplasmic loop connecting helices IV and V of the melibiose permease from Escherichia coli is involved in the process of Na+-coupled sugar translocation.
J Biol Chem. 2003 Jan 17;278(3):1518-24. Epub 2002 Nov 5., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC8 p.Val343Gly Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Login to comment 7 ABCC8 p.Val343Gly Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. Login to comment 73 ABCC8 p.Val343Gly Identification of R149C/V343G Second-site Revertants-A strategy developed by Ding and Wilson was used (28). Login to comment 83 ABCC8 p.Val343Gly ABCC8 p.Val343Gly The second-site mutation present in helix X (V343G) of the revertant R149C/V343G described below is indicated in bold face. Login to comment 104 ABCC8 p.Val343Gly TABLE I NPG binding constants of the C-less permease, of the permease carrying different substitutions at position 141 or 149, and of the second-site revertant R149C/V343G All RSO membrane vesicles harboring the C-less or MelB mutants were prepared and concentrated (15-25 mg of protein/ml) in Na- -free, 0.1 M potassium phosphate buffer (pH 7) containing carbonyl cyanide m-chlorophenylhydrazone (5 ␮M) and monensin (0.75 ␮M) as described in the Fig. 3 legend. Login to comment 111 ABCC8 p.Val343Gly ABCC8 p.Val343Gly Mutant Location Bmax (nmoles/mg protein) KD KNa ␮M mM C-less 0.6 0.6 0.6 R141C 0.2 2.5 0.1-0.5 R141K Loop 4-5 0.2 1.7 0.3 R141Q 0.3 3.1 0.3 R149C NS NS NS R149K Loop 4-5 0.3 7.7 0.3 R149Q 0.3 7.3 0.4 R149C/V343G Loop 4-5/TM X 0.2 1.7 0.2 V343G TM X 0.3 4.4 1.6 FIG. 3. Login to comment 168 ABCC8 p.Val343Gly Fig. 6 shows that R149C/V343G RSO membrane vesicles accumulated melibiose up to 10% of the levels observed in C-less MelB vesicles. Login to comment 170 ABCC8 p.Val343Gly Remarkably, although R149C/V343G had limited capacity to concentrate the substrates, it displayed sugar affinity and Naϩ activation constants very close or identical to those of C-less MelB (Table I). Login to comment 172 ABCC8 p.Val343Gly One may finally note that the double mutant has a comparatively better affinity for ␣-galactosides (KD) or for Naϩ ions on the basis of the KNa value than any of the individual single R149C or V343G mutants (Table I). Login to comment 173 ABCC8 p.Val343Gly Finally, the effects of various thiol reagents on the transport and sugar binding properties of R149C/V343G RSO membrane vesicles were analyzed. Login to comment 177 ABCC8 p.Val343Gly To identify the step of the R149C/V343G transport cycle inhibited by MTSETϩ inhibited, we analyzed the effect of this reagent on the ␣-[3 H]NPG binding to RSO membrane vesicles and looked for an eventual protection by the co-substrates (Fig. 7). Login to comment 182 ABCC8 p.Val343Gly Inhibition of Na؉ -dependent sugar transport of R149C/ V343G in RSO membrane vesicles by MTSET؉ . Login to comment 188 ABCC8 p.Val343Gly Selective protection against inactivation by MTSET؉ of ␣-[3 H]NPG binding to R149C/V343G RSO membrane vesicles by the sugar. Login to comment 224 ABCC8 p.Val343Gly Analysis of the properties of the second-site suppressor mutant R149C/V343G provided further clues to the mechanism by which Arg-149 participates in the process of sugar binding. Login to comment 225 ABCC8 p.Val343Gly The presence of an additional mutation (V343G) led to partial recovery of transport activity despite the absence of the native arginine at position 149. Login to comment 230 ABCC8 p.Val343Gly The observed inhibition of both R149C/V343G sugar transport and binding activity by MTSETϩ or PCMBS- provides insight into the relative localization of Arg-149 with respect to the sugar binding site. Login to comment