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

Abstract

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...

Proteomic Analysis of the Mode of Antibacterial Action of Silver Nanoparticles

“…Figure 3A shows that three protein bands were detected on the 1D immunoblot probed with polyclonal antibodies raised against the OmpA protein. The observed expression pattern was very similar to those reported previously: [27][28][29][30][31][32] mature OmpA appears in both heat-modified (35 kDa) and unmodified forms (30 kDa), whereas proOmpA appears in a higher molecular weight form (37 kDa) on SDS-PAGE. In nano-Ag treated cells, the expression of the 37 kDa band, corresponding to proOmpA, was markedly enhanced.…”

“…In the absence of membrane potential or ATP, the precursor proteins are not incorporated into the inner membrane, and the signal sequences are not cleaved. 27,28,[31][32][33][34][35] Consequently, the precursor proteins accumulate in the cytoplasm under these conditions. We reasoned that the accumulation of envelope proteins precursors in nano-Ag treated cells was due to the effects of that nano-Ag had on the bacterial membrane potential and/or ATP levels.…”

“…It is also noteworthy that the outer membrane protein precursors are known to have extremely short cellular half-lifes (3-5 s), 31 and are not normally detected under normal conditions. [27][28][29][30][31][32][33] This was the case for the untreated samples in our experiments (Figure 2B and 3B).…”

“…It is also noteworthy that the outer membrane protein precursors are known to have extremely short cellular half-lifes (3-5 s), 31 and are not normally detected under normal conditions. [27][28][29][30][31][32][33] This was the case for the untreated samples in our experiments (Figure 2B and 3B). Thus, our proteomic analysis and immunoblot assays demonstrated that nano-Ag treatment resulted in an accumulation of precursor forms of OmpA, apparently due to an inhibition of their processing into shorter, mature forms.…”

A unifying mechanism for protein transport through the core bacterial Sec machinery

“…By contrast, secretion in bacteria is usually, but not exclusively, post-translational, i.e. not coupled to protein synthesis [15][16][17][18]. However, even 'post-translational' transport can start before translation is complete [19], and indeed the early targeting steps are probably initiated co-translationally [20].…”