Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.
We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.
Laboratory of Molecular Biology, University of Wisconsin-Madison 53706, USA.
P
P
Blount
S I
SI
Sukharev
P C
PC
Moe
M J
MJ
Schroeder
H R
HR
Guy
C
C
Kung
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000737:chemistry; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006639:Histidine / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D007473:Ion Channels; D008081:Liposomes; C086596:MscL protein, E coli; D006639:Histidine; D015245:Deoxyribonuclease BamHI; D000469:Alkaline Phosphatase