Intragenic suppression of gain-of-function mutations in the Escherichia coli mechanosensitive channel, MscL.
Mechanosensitive channels play an important role in protecting bacterial cells from osmotic downshock by serving as biological 'pressure release valves'. One of these channels, MscL, is found throughout the bacterial kingdom, but has been most studied in Escherichia coli. The E. coli MscL is a 136-amino-acid protein organized as a homopentamer with each subunit containing two transmembrane segments. Previous studies have shown that several residues, including V23 and G26, are essential for normal function of MscL; very severe gain-of-function phenotypes in which cell growth slows or is arrested can result from residue substitutions at these positions. Through random mutagenesis and growth selection, we have generated intragenic suppressors of the V23A and G26S mutations. The suppressor mutants have been characterized by growth phenotype, Western blot and patch clamp. Most of the mutations that render phenotypic suppression are located in the transmembrane domains with additional sites lying in the periplasmic loop. In contrast, only one mutation is found in the amino-terminal S1 domain, and none is found within the carboxyl-terminal domain. Not only have these findings revealed functional domains and subdomains critical for MscL function, but they also predict a pair of residues that interact directly during channel opening.
Li
Yuezhou
Y
Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9040, USA.
Wray
Robin
R
Blount
Paul
P
D000222:Adaptation, Physiological / Q000235:genetics; D019943:Amino Acid Substitution; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000502:physiology*; D007473:Ion Channels / Q000235:genetics* / Q000502:physiology*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013489:Suppression, Genetic
D029968:Escherichia coli Proteins; D007473:Ion Channels; D008565:Membrane Proteins; C086596:MscL protein, E coli