CROSS-LINKING STUDIES AND MEMBRANE LOCALIZATION AND ASSEMBLY OF RADIOLABELED LARGE MECHANOSENSITIVE ION-CHANNEL (MSCL) OF ESCHERICHIA-COLI

The gene encoding the large conductance mechanosensitive ion channel ( MscL) of Escherichia coli and several deletion mutants of mscL were cl oned under the control of the T7 RNA polymerase promoter. Transformati on of these constructs into an E. coli strain carrying an inducible T7 RNA polymerase gene allowed the specific production and labelling of MscL with [S-35]methionine. Preparation of membrane fractions of E. co li cells by sucrose gradient centrifugation indicated that the radiola belled MscL was present in the inner cytoplasmic membrane in agreement with results of several studies. However, treatment of the labelled c ells and cell membrane vesicles with various cross-linkers resulted in the majority of labelled protein migrating as a monomer with a small proportion of molecules (approximate to 25%) migrating as dimers and h igher order multimers. This result is in contrast with a finding of a study suggesting that the channel exclusively forms hexamers in the ce ll membrane off. coli (1) and therefore may have profound implication for the activation and/or ''multimerization'' of the channel by mechan ical stress exerted to the membrane. In addition, from the specific ac tivity of the radiolabelled protein and the amount of protein in the c ytoplasmic membrane fraction we estimated the number of MscL ion chann els expressed under these conditions to be approximately 50 channels p er single bacterium. (C) 1997 Academic Press.