Referenced in: none

Automatically associated channels: Kv2.1 , Slo1

Title: Assembly pathway of newly synthesized LamB protein an outer membrane protein of Escherichia coli K-12.

Authors: G H Vos-Scheperkeuter, B Witholt

Journal, date & volume: J. Mol. Biol., 1984 Jun 5 , 175, 511-28

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/6204059

Abstract
The assembly of newly induced LamB protein (phage lambda receptor) was investigated in an operon fusion strain of Escherichia 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 two distinct antisera directed against detergent-solubilized LamB trimers and completely denatured LamB monomers, respectively. Anti-trimer antibodies recognized both monomers and trimers, whereas anti-monomer antibodies only reacted with monomers. Provided appropriate solubilization conditions were used, both antisera were able to immunoprecipitate intracellular mature LamB protein quantitatively. Following induction, the first LamB antigenic determinants were detected after 60 to 80 seconds; 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 to 50 seconds with respect to their synthesis. Therefore, either 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 was found to occur in at least three clearly distinguishable states. State I is the LamB monomer, state II corresponds to a metastable trimer that dissociates in sodium dodecyl sulphate above 60 degrees C, and state III is the state LamB trimer that dissociates in sodium dodecyl sulphate only at temperatures above 90 degrees C. The chase kinetics of these states showed that conversion of newly synthesized LamB monomers to stable LamB trimers occurred in two stages: state I monomers were chased into metastable state II trimers rapidly (t 1/2 = 20 s), whereas stabilization of state II trimers to state III trimers was a relatively slow (t 1/2 = 5.7 min) process. Based on our results, a timing sequence in the assembly of outer membrane LamB protein is proposed.