A Site-specific, Membrane-dependent Cleavage Event Defines the Membrane Binding Domain of FtsY

Abstract

Targeting of many polytopic proteins to the inner membrane of prokaryotes occurs via an essential signal recognition particle-like pathway. Unlike the general secretory pathway, the proteins involved in this pathway and their activities appear in many respects to mirror closely those of their eukaryotic homologues. However, the Escherichia coli signal recognition particle receptor, FtsY, differs significantly at the amino terminus from the eukaryote homologue ␣-subunit of the signal recognition particle receptor. In addition, there is no prokaryote homologue of the transmembrane ␤-subunit of the receptor. Therefore, FtsY must assemble on the membrane in a unique manner. Using assays designed to accurately discriminate membrane-bound proteins from aggregated material, we found that in contrast to a previous report, only amino acids 1-284 of FtsY are necessary and sufficient for membrane assembly. These amino acids together constitute a bona fide membrane binding domain that includes both the regions originally designated A and N based on sequence comparisons. Furthermore, we found that a membranebound factor mediates specific cleavage of some membrane-bound FtsY molecules between the N and G regions previously believed to be functionally linked to generate a novel membrane-bound isoform composed of only the AN domain.

Escherichia coli Signal Recognition Particle Receptor FtsY Contains an Essential and Autonomous Membrane-binding Amphipathic Helix

“…20). This proposal has been confirmed several times by demonstrating that FtsY assembles on membranes via interactions with a trypsin-sensitive component (22,37). Finally, the most compelling evidence for FtsYmembrane protein association has recently been offered by studies of the interaction between FtsY and the SecYEG complex in E. coli (11,12).…”

“…Nevertheless, various studies have suggested that FtsY functions as a membrane-bound receptor (18,19). Interestingly, A-domain-truncated FtsY versions exhibit strong affinity for membrane lipids (20,21), possibly through the N-domain (22), an interaction that seems to be dominated by electrostatic forces (21). However, the precise lipid-interacting domain in FtsY has not been defined, and the mechanism and functional role of lipid binding to the receptor remain elusive.…”

Identification of the Preprotein Binding Domain of SecA

“…Membrane Flotation Experiments-Ultracentrifugal sedimentation experiments were carried out in a bench-top ultracentrifuge (TLX120 Optima; TLA120.2 rotor; Beckman), using polypropylene tubes (0.2 ml) as described (34,35). Reactions (15 l in buffer B (50 mM Tris-Cl, pH 8, 50 mM KCl, 5 mM MgCl 2 )) containing SecA or SecA⌬Bulb (0.5 g) in the presence or absence of inner membrane vesicles (IMVs) (Յ3 g) and proOmpA-His (0.12 g) (36) were adjusted to 1.74 M final sucrose concentration and deposited at the bottom of the tube.…”

“…SecA PBD Mutants Bind to SecYEG-containing Membranes-To examine the effect of PBD mutations on the ability of SecA to bind to SecYEG and to form the translocase ternary complex, we employed membrane flotation (34,35). Inverted IMVs, treated with urea to remove endogenous SecA (19), were loaded at the bottom of a sucrose gradient (corresponding to lanes 7-9 in Fig.…”

X‐ray structure of the T. Aquaticus Ftsy:GDP complex suggests functional roles for the C‐terminal helix of the SRP GTPases

“…58 The structural and functional implications of these interactions have recently been discussed 56 -in the intact FtsY they may couple the putative targeting function of the N-terminal A-domain (or perhaps, the N1 helix itself) to the NG GTPase domain. 12,26,59 That a similar orientation of the C-terminal helix is maintained in the structure of T. aquaticus FtsY, despite the first 20 amino acids of the NG domain being absent (see above), is explained by a crystal packing interaction that compensates-although much of the linker peptide between the N-and G-domains is disordered, residues 83-94 can be located unambiguously in the electron density map, splayed across a crystal contact to pack at the N/ G interface of an adjacent monomer [ Fig. 3(B)].…”