Two-stage Binding of SecA to the Bacterial Translocon Regulates Ribosome-Translocon Interaction

Abstract

The bacterial translocon interacts with both SecAbound preproteins and nascent chain-ribosome complexes during Sec and signal recognition particle-dependent protein translocation, respectively. In their inactive state, translocons are saturated with ribosomes and SecA protein, reflecting the inherent affinity of these components for one another. We found that SecA and ribosomes are bound simultaneously and noncompetitively to a common set of inactive translocons. Furthermore, we demonstrate that at a later stage in binding, SecA possesses a ribosome-translocon dissociation activity that is coupled to its ATP-dependent membrane insertion and retraction cycle that drives protein translocation. This novel activity is presumably important in the commitment of the translocon to the Sec-dependent pathway. These results also provide a rationale for the compatibility and regulation of multiple protein translocation pathways that each makes distinct demands on a common translocon core.

Role of a Conserved Glutamate Residue in the Escherichia coli SecA ATPase Mechanism

“…As expected from previous studies, wild type SecA ATPase activity is stimulated about 10-fold in the presence of SecYEG/membrane and pre-protein ( Fig. 2A) (22). A more sensitive and accurate radiometric assay used to measure the steady-state rate constants for wild type SecA (k cat ϭ 0.2-0.3 s Ϫ1 ) and mutant SecA endogenous ATPase activity indicates that SecA-E210D retains some level of activity (k cat ϭ 0.15 s Ϫ1 ), but SecA-E210A mutant activity is near background level (k cat ϭ 0.04 s Ϫ1 ) (Fig.…”

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

“…14,15 Similarly, bacterial ribosomes have been shown to specifically bind to SecYEG complexes, 20,21 although the physiological significance of this finding remains the subject of investigation. In Archaea, the nature of the ribosomal receptor had, until now, not been defined.…”

“…19 Moreover, the ability of bacterial ribosomes to bind to the bacterial homologue of the Sec61-based translocon, the SecYEG complex, has been shown. 20,21 Indeed, ribosome binding to Sec complexes in both domains of Life occurs with comparable dissociation constants.…”

In Vivo Membrane Topology of Escherichia coli SecA ATPase Reveals Extensive Periplasmic Exposure of Multiple Functionally Important Domains Clustering on One Face of SecA

“…S1). Although the observed increase in specific activity of SecA labeling appears to be lower than the increase in SecYEG overproduction, we have shown previously that only a fraction of overproduced SecYEG protein properly assembles in the membrane where it gives rise to an increase in SecA high affinity binding sites and SecA-dependent translocation ATPase activity (42). However, we cannot rule out the possibility that some of the MPB-labeled residues of SecA arose from periplasmic exposure of a phospholipid-bound pool of SecA, although such speculation is inconsistent with the extractability of this pool of SecA by reagents that classically remove only peripherally bound membrane proteins (15).…”