Membrane Targeting of Ribosomes and Their Release Require Distinct and Separable Functions of FtsY

Abstract

The mechanism underlying the interaction of the Escherichia coli signal recognition particle (SRP) receptor FtsY with the cytoplasmic membrane is not fully understood. We investigated this issue by utilizing active (NG؉1) and inactive (NG) mutants of FtsY. In solution, the mutants comparably bind and hydrolyze nucleotides and associate with SRP. In contrast, a major difference was observed in the cellular distribution of NG and NG؉1. Unlike NG؉1, which distributes almost as the wild-type receptor, the inactive NG mutant accumulates on the membrane, together with ribosomes and SRP. The results suggest that NG function is compromised only at a later stage of the targeting pathway and that despite their identical behavior in solution, the membrane-bound NG-SRP complex is less active than NG؉1-SRP. This notion is strongly supported by the observation that lipids stimulate the GTPase activity of NG؉1-SRP, whereas no stimulation is observed with NG-SRP. In conclusion, we propose that the SRP receptor has two distinct and separable roles in (i) mediating membrane targeting and docking of ribosomes and (ii) promoting their productive release from the docking site.Membrane-bound ribosomes are responsible for the biosynthesis of many integral membrane proteins that insert into the membrane in a co-translational manner (1, 2). Targeting of these ribosomes to the cytoplasmic membrane in Escherichia coli requires the signal recognition particle (SRP) 2 receptor, FtsY (3). In addition, under FtsY depletion conditions, the expression of polytopic membrane proteins such as LacY (4), SecY (3), and MdfA 3 is repressed. Besides its interaction with the inner membrane, FtsY functionally interacts with the SRP and the SRP protein Ffh in a nucleotide-dependent manner (reviewed in Ref. 5). Moreover, additional studies demonstrated that FtsY forms a complex with membrane-bound ribosomes (6) and the SecYEG translocon (7, 8). These observations thus underscore the central role of the SRP receptor in ribosome targeting and biogenesis of membrane proteins (1, 9). However, despite extensive genetic, biochemical, and structural studies, important aspects of the function of FtsY are not yet fully understood.At steady state, FtsY is distributed between the cytoplasm and the membrane (10), but the cytosolic form does not seem to be essential for membrane protein biogenesis or cell survival (11). In contrast, various studies suggest that FtsY functions as a membrane-bound receptor (11, 12), in agreement with observations that it interacts with membrane-bound ribosomes (6) and the translocon (7,8). Interestingly, however, FtsY has no known membrane anchor partner homologous to the mammalian ␤-subunit of the SRP receptor.FtsY contains three domains. The C-terminal N-and G-domains (together 302 residues long) constitute a universally conserved SRP-GTPase (13) that interacts with the homologous NG domain of the SRP protein Ffh. In its N terminus, FtsY contains an acidic A-domain (195 residues long) that was proposed to mediate membrane targeting and...

Anionic Phospholipids and the Albino3 Translocase Activate Signal Recognition Particle-Receptor Interaction during Light-harvesting Chlorophyll a/b-binding Protein Targeting

“…2A, green residues (40 -42)). Importantly, one phenylalanine in E. coli FtsY and two phenylalanines in cpFtsY preceding this motif play critical roles in inducing helix formation in this motif and are hence required for membrane binding of these receptors (40,42,48). Consistent with these results, we found that truncation of the N-terminal residues of cpFtsY (to yield cpFtsY-NG) abolished the PG-induced stimulation of GTPase assembly (Fig.…”

Lipids Trigger a Conformational Switch That Regulates Signal Recognition Particle (SRP)-mediated Protein Targeting

“…Only FtsY variants that contain at least a minimal MTS are functional in vivo and can be activated in GTP hydrolysis upon SRP interaction (13). In the crystal structure of the SRP-FtsY GTPase heterodimer, ␣N1 of the N domain is absent in both proteins (33,34).…”

“…However, the A domain is not essential in Escherichia coli as a truncation variant (termed NGϩ1) is functional in vivo (11). It was shown that the FtsY GTPase is activated by anionic phospholipids (9) and that the membrane interaction of FtsY is crucial for the release of RNCs from the SRP-FtsY complex (12,13), which was confirmed recently (14). Membrane interaction of E. coli FtsY depends on a conserved motif at the N terminus of the N domain, referred to as membrane-targeting sequence (MTS) (15).…”

The Membrane-binding Motif of the Chloroplast Signal Recognition Particle Receptor (cpFtsY) Regulates GTPase Activity

“…Constructs were labeled with ratios of labeled and unlabeled Met such that equal 35 35 S signal from a given protein band as analyzed by SDS-PAGE and phosphorimaging. Equimolar amounts of proteins were added to each experiment (supplemental Fig.…”