RESUMO
A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.
Assuntos
Proteínas de Escherichia coli/metabolismo , Lipídeo A/química , Proteínas de Membrana/metabolismo , Partícula de Reconhecimento de Sinal/química , Proteínas da Membrana Bacteriana Externa/química , Sistema Livre de Células , Diglicerídeos/química , Escherichia coli/metabolismo , Lipídeos/química , Lipossomos/química , Modelos Moleculares , Peptídeos/química , Transporte Proteico , Proteolipídeos/química , Canais de Translocação SECRESUMO
The spc operon of Escherichia coli encodes 11 ribosomal proteins and SecY. The secY gene and downstream rpmJ encoding a ribosomal protein, L36, are located distal to the promoter of the spc operon. It has been suggested that the stability of SecY mRNA depends on rpmJ unless a rho-independent terminator is inserted immediately downstream of secY. Moreover, it has been suggested that RpmJ is dispensable for E. coli. We constructed rpmJ null strains, AY101 (DeltarpmJ::tetA) and AY201 (DeltarpmJ::cat), by replacing rpmJ with tetA, which encodes a membrane protein responsible for tetracycline-resistance, and cat, which encodes a cytoplasmic chloramphenicol acetyltransferase, respectively. Depletion of RpmJ did not inhibit protein synthesis, whereas the growth of AY101 was defective at high temperatures. The level of SecY mRNA decreased significantly in both disruptants even though the rho-independent terminator was inserted immediately downstream of secY. Some periplasmic proteins were missing in the disruptants with a concomitant increase in the amount of phage shock protein in the inner membrane. These phenotypes caused by the rpmJ null mutation were corrected by a plasmid carrying secY, but not by one carrying rpmJ.
