Protein translocationin vitro: Biochemical characterization of genetically defined translocation components

Abstract

Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation in E. coli. The powerful combination of these approaches is exemplified in the use of an in vitro protein synthesis-protein translocation system to analyze the role of genetically defined components of the protein translocation machinery. We describe in this review recent results focusing on the function of the secA, secB, and secY gene products and the demonstration of their requirement for in vitro protein translocation. The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase. We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or "protein-ATPase."

A Significant Fraction of Functional SecA Is Permanently Embedded in the Membrane

“…2 suggest that release of SecA is not related to protein translocation. To investigate further the relations between SecA release and protein translocation, we examined the kinetics of release of the membrane-associated 35 S-SecA [1][2][3][4][5] or 90 l of translocation buffer (ϪpOmpA) with (lanes 9 -13) or without (lanes 7 and 8) the energy source (ATP), followed by incubation for 15 min at 37°C or 0°C as indicated. SecA (20 g) was present in lanes 4, 5, 12, and 13.…”

“…S-pOmpA. A, the reconstituted membranes were added to translocation buffer containing the energy source with (ϩ pOmpA, lanes [1][2][3][4][5][6] or without (Ϫ pOmpA, lanes 8 -13) 35 S-pOmpA. After incubation at 37°C for the times indicated, the membranes were isolated immediately by centrifugation without proteinase K treatment (lanes 1-4 and 8 -11) or after proteinase K (0.5 mg/ml) treatment in the absence (lanes 5 and 12) or presence (lanes 6 and 13) of 1% Triton X-100 (TX-100).…”

“…Translocation of most periplasmic and outer membrane proteins across the cytoplasmic membrane of Escherichia coli occurs through the Sec-dependent pathway (1,2), which consists of a distinct set of Sec proteins (3,4). SecA plays a pivotal role in protein translocation.…”

“…On the other hand, extraction experiments with urea or Na 2 CO 3 suggested that a significant fraction of SecA behaves like an integral membrane protein (30). 1 Recently, Kim et al (33) reported that a strain harboring a plasmid containing the secD secF locus possesses SecA almost entirely as an integral membrane form and displays normal protein translocation as measured by rapid processing of preproteins in vivo (although it is possible that only a small fraction of SecA is active which accounts for the apparent "normal" translocation). Kim et al (33) suggested that the integral SecA is the catalytically active form in vivo and that, in this state, it might form a part of the reported protein-conducting channels (34).…”

Translocation of the precursor of α‐amylase into Bacillus subtilis membrane vesicles

“…A major advance has been the development of an in vitro protein translocation system based on everted inner membrane vesicles [25] and, more recently, by the use of purified preproteins and translocase components reconstituted into liposomes [26,27]. Despite many attempts [28], it has not yet been possible to obtain a reliable in vitro translocation system for B. subtilis.…”