No abstract

Flagella are well characterized as the organelles of locomotion and allow bacteria to react to environmental changes. The assembly of flagella is a multistep process and relies on a complex type III export machinery located in the cytoplasmic membrane. The FlhF protein is essential for the placement and assembly of polar flagella and has been classified as a signal-recognition particle (SRP)-type GTPase. SRP GTPases appeared early in evolution and form a unique subfamily within the guanine nucleotide binding proteins with only three members: the signal sequence-binding protein SRP54, the SRP receptor FtsY, and FlhF. We report the crystal structures of FlhF from Bacillus subtilis in complex with GTP and GMPPNP. FlhF shares SRP GTPase-specific features such as the presence of an N-terminal ␣-helical domain and the I-box insertion. It forms a symmetric homodimer sequestering a composite active site that contains two head-to-tail arranged nucleotides similar to the heterodimeric SRP-targeting complex. However, significant differences to the GTPases of SRP and the SRP receptor include the formation of a stable homodimer with GTP as well as severe modifications and even the absence of motifs involved in regulation of the other two SRP GTPases. Our results provide insights into SRP GTPases and their roles in two fundamentally different protein-targeting routes that both rely on efficient protein delivery to a secretion channel.flagellum ͉ protein translocation ͉ x-ray structure analysis ͉ protein targeting

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.The biogenesis of most membrane proteins and many secretory proteins depends on the signal recognition particle (SRP) 4 and the SRP receptor (SR). SRP binds to N-terminal signal sequences of nascent polypeptide chains at the ribosome (ribosome-nascent chain complexes (RNCs)) and acts as an adaptor between the ribosome and the membrane-embedded translocation channel (1-3). Interaction of SRP with SR (FtsY in bacteria and archaea and SR␣ in eukarya) specifies the target membrane and allows for the precise coordination of RNC release from SRP and its transfer to the translocation channel. Protein targeting critically depends on the two homologous GTPases present in SRP and SR forming a heterodimer (4). GTP binding to SRP and SR is required for heterodimer formation and GTP hydrolysis triggers the dissociation of the SRP-SR complex which resets the SRP system for a new round of translocation (5). Although FtsY does not contain a hydrophobic, transmembrane sequence, it was shown to be almost exclusively localized at the membrane (6). FtsY contains three domains: an N-terminal negatively charged A domain of unknown structure and function and the highly conserved N and G domains that form a structural and functional unit, the NG domain (7, 8) (see Fig. 1A). The A domain acts as negative regulator of the FtsY GTPase in a lipid-free environment (9) and was suggested to participate in membrane interaction of FtsY by its N-terminal region (10). 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). Recently, strong genetic ...

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...

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