ASSEMBLY PATHWAY OF NEWLY SYNTHESIZED LAMB PROTEIN AN OUTER-MEMBRANE PROTEIN OF ESCHERICHIA-COLI K-12

[摘要] The assembly of newly induced LamB protein (phage .lambda. receptor) was investigated in an operon fusion strain of E. coli, in which the lamB gene is expressed under lac promoter control. The induction kinetics both for total cellular and for cell surface-exposed LamB protein were studied by immunochemical detection methods, using 2 distinct antisera directed against detergent-solubilized LamB trimers and completely denatured LamB monomers, respectively. Anti-trimer antibodies recognized both monomers and trimers. Anti-monomer antibodies only reacted with monomers. Provided appropriate solubilization conditions were used, antisera were able to immunoprecipitate intracellular mature LamB protein quantitatively. Following induction, the 1st LamB antigenic determinants were detected after 60-80 s; detection of the newly synthesized protein by anti-monomer antibodies slightly preceded that by anti-trimer antibodies, a finding that could be partly explained by the observation that anti-monomer antibodies recognized a larger fraction of nascent LamB than did anti-trimer antibodies. Exposure of antigenic determinants at the cell surface was delayed for 30-50 s with respect to their synthesis. Thus, translocation or conformational changes must be rate-limiting in the series of processes that eventually convert the newly synthesized protein into its mature outer membrane state. LamB protein occurred in at least 3 clearly distinguishable states. State I is the LamB monomer, state II corresponds to a metastable trimer that dissociates in sodium dodecylsulfate > 60.degree. C and state III is the state LamB trimer that dissociates in sodium dodecylsulfate only at temperatures > 90.degree. C. The chase kinetics of these states showed that conversion of newly synthesized LamB monomers to stable LamB trimers occurred in 2 stages: state I monomers were chased into metastable state II trimers rapidly (t1/2 = 20 s). Stabilization of state II trimers to state III trimers was a relatively slow (t1/2 = 5.7 min) process. A timing sequence in the assembly of outer membrane LamB protein is proposed.