Precursor Proteins Are Intermediates <i>in vivo</i>, in the Synthesis of Two Major Outer Membrane Proteins, the OmpA and OmpF Proteins, of <i>Escherichia coli</i> K12

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The rate of synthesis of the OmpA and OmpF proteins, two of the major outer membrane proteins of E.sckerichia coli K12, was determined. At 25 "C both proteins were translated at 6.5 amino acids/s, and the OmpF protein was translated at 15 amino acids/s at 37 "C. The former rate corresponded to a synthesis time of just over SO s for both proteins, which is significantly faster than their reported rates of assembly into the outer membrane at 25 "C. The kinetics of processing of the pro-OmpF protein were also investigated in detail, and the pro-OmpF half-life estimated to be 3 -5 s at 25 "C. However a fraction of the precursor was processed more slowly, which may explain the discrepancy between these data and our earlier published estimate of 30 s. Pro-OnipA protein was processed with similar kinetics. These results demonstrate that the rate-limiting step in the assembly of both proteins into the outer membrane is post-translational and follows the processing step.The outer membrane proteins of gram-negative bacteria share a number of biosynthetic steps with two other classes of exported protein, the inner membrane and periplasmic proteins. These include: translation, export (i.e. transfer across the inner membrane) and processing (for proteins synthesized initially in precursor form). In addition, outer membrane proteins must be translocated to the outer membrane. Despite these similarities the rate of assembly of outer membrane proteins is two or three times slower than that of inner membrane proteins (though perhaps faster than that of periplasmic proteins) at 25-30 "C [I -41. Intuitively it would seem that the rate-limiting step in outer membrane protein assembly must be either translocation (the only step unique to outer membrane proteins) or processing (which should not affect the kinetics of inner membrane protein assembly). In an earlier paper [ S ] we reported that newly synthesized pro-OmpF protein had a relatively long half-life of 30 s, suggesting that the processing reaction might be ratelimiting for assembly. However, we also found that newly synthesized, processed molecules of both OmpF and OmpA proteins were present in the inner membrane after a 10-s pulse ; these results suggest that translocation may be rate-These results are at odds with those of Lin and Wu [2], who have suggested that the rate-limiting step in outer membrane protein assembly is translation of the niRNA. However, their evidence is indirect and not in accordance with the data of Boyd and Holland [6], who could detect no difference between the 'run-out' times (a crude measure of the translation rate) of the OmpF protein and a representative cytoplasmic protein in Escherichia coli Bjr.In view of these problems we decided to reinvestigate the kinetics of processing of the OmpF and OmpA proteins and to measure directly their rates of synthesis. Our results indicate that neither processing nor translation are slow enough to permit them to be considered rate-limiting. Since the only steps to follow processing are translocation and irre...

Section: Antiserumentioning

confidence: 89%

Section: Antiserumentioning

confidence: 99%

Section: Irnrnunoprecipitationmentioning

confidence: 99%

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Rate of Translation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer‐Membrane Proteins, the OmpA and OmpF Proteins, of Escherichia coli K12

Crowlesmith¹,

Gamon²

1982

Self Cite

“…Thirdly, the possible loss of OmpA fragments into the medium could indicate the absence of stop-transfer [40,41] or dissociation sequences [38]. This last possibility seems somewhat less likely because it would indicate that the polypeptide is inserted into the outer membrane during synthesis, and this is apparently not the case [42]. Whichever of these possibilities is correct remains to be elucidated.…”

Section: Stable and Unstable Translation Productsmentioning

confidence: 86%

Export of a Protein into the Outer Membrane of Escherichia coli K12

Bremer

Cole

Hindennach

et al. 1982

The cloned ompA gene encoding the major outer membrane protein OmpA of Eschevichiu coli has been shortened in vitro by exonuclease digestion from the end corresponding to the CO2H terminus of the protein. Nine derivatives were identified which still possessed sub:tantial parts of the ompA gene and one was constructed which had suffered a small deletion early in the gene. Gene fragments encoding NH2‐terminal OmpA sequences of 45, 133, 193, and 227 residues of the 325 amino acids of OmpA were examined in detail at the DNA level and for OmpA protein fragments synthesized. The latter two fragments were incorporated into the outer membrane and all known functions of the OmpA protein were expressed whereas the fragment with 133 OmpA‐specific residues was not stably incorporated into this membrane. In all cases where OmpA functions were observed, an OmpA‐specific polypeptide of Mr24000 was found in cell envelopes, regardless of the size of the residual ompA sequences and of the fused coding sequences in the vector DNA. Pulse‐label experiments revealed larger initial translation products, most of which were degraded to the protein of Mr 24000. The 133‐residue OmpA fragment was also detected but proved to be entirely unstable. It is argued that the OmpA protein consists of two domains and that the NH2‐terminal moiety from residues 1 to about 180 represents the membrane domain of the polypeptide. Therefore, the loss of about 50,possibly less, CO2H‐terminal residues from this domain suffices to interfere with stable incorporation into the outer membrane.

“…The transfer may occur via adhesion sites as proposed by Smit and Nikaido [39] and could explain the isolation of differentiated membrane fractions containing various components of the outer membrane [40]. The detection of a mature form of OmpF in a detergent-soluble fraction during pulse-chase experiments could be taken as evidence for this step [15]. The integration of OmpF into the outer membrane would finally expose the MoF3 site.…”

Section: Discussionmentioning

confidence: 99%

Assembly of the OmpF porin of Escherichia coli B

Pagès

Bolla

1988

The different conformations of the outer membrane protein OmpF of Escherichia coli B were studied with immunological probes. The antigenic determinants recognized by one monoclonal (MoF3) and two polyclonal antibodies were investigated under various conditions of solubilization which modify the association of OmpF with other membrane components, such as lipopolysaccharide. Several polymeric forms of the protein could be detected after extraction at 37°C or 56°C. The monoclonal antibody, which is specific to an exposed region of native OmpF, recognized various trimeric forms in an immunoprecipitation assay. Under the same conditions, the binding of polyclonal antibodies apparently induced strong conformational rearrangements, since the pattern of trimeric forms detected was greatly modified. The conversion of newly synthesized monomers of OmpF to the various trimer forms was investigated using these antibodies. The trimerization occurred rapidly but the appearance of the native conformation of OmpF was delayed. Some additional step was required to expose the MoF3-specific antigenic site at the surface of the trimeric form. These results are discussed in relation to the structure of OmpF and its association with lipopolysaccharide in the outer membrane.OmpC and OmpF are the major outer membrane proteins of Escherichia coli K12, functioning as passive diffusion pores for small hydrophilic molecules [l -31. These porins are assembled as trimer units at the cell surface [4 -61. The structural genes and the sequence of these proteins have been previously described and the regulation of their syntheses is being intensively studied [l, 21. In E. coli B, only one outer membrane porin, related to OmpF, is present. This protein is identical to the E. coli K12 porin except for three substitutions, at amino acid positions 66, 117 and 262 [7]. The assembly of outer membrane proteins is an intriguing and complex process requiring several steps in various cellular compartments during the export process. During and after synthesis the polypeptide chains are translocated across the cytoplasmic membrane where the cleavage of the signal sequence occurs. Several factors are required during this translocation as demonstrated by genetic and biochemical studies [8 -101. The importance of membrane fluidity, the energy requirement and the kinetics of processing are now well documented for this step.Later steps, which ensure the correct insertion of the proteins into the outer membrane, are less well understood. In the case of the outer membrane protein OmpA, an intermediate form (imp-OmpA) is detected, just after processing, at the external side of the cytoplasmic membrane [ll]. The binding of lipopolysaccharide may induce a conformational change promoting the subsequent integration into, the outer membrane. However, for OmpA neither dimeric nor trimeric assembly is required for the integration of the native protein.The maltoporin LamB [ 121 undergoes several conformational changes in order to achieve its native trimeric form in the Corr...