Structural basis of signal sequence surveillance and selection by the SRP–FtsY complex

Signal-recognition particle (SRP)-dependent targeting of translating ribosomes to membranes is a multistep quality-control process. Ribosomes that are translating weakly hydrophobic signal sequences can be rejected from the targeting reaction even after they are bound to the SRP. Here we show that the early complex, formed by Escherichia coli SRP and its receptor FtsY with ribosomes translating the incorrect cargo EspP, is unstable and rearranges inefficiently into subsequent conformational states, such that FtsY dissociation is favored over successful targeting. The N-terminal extension of EspP is responsible for these defects in the early targeting complex. The cryo-electron microscopy structure of this 'false' early complex with EspP revealed an ordered M domain of SRP protein Ffh making two ribosomal contacts, and the NG domains of Ffh and FtsY forming a distorted, flexible heterodimer. Our results provide a structural basis for SRP-mediated signal-sequence selection during recruitment of the SRP receptor

Structures of the Signal Recognition Particle Receptor from the Archaeon Pyrococcus furiosus: Implications for the Targeting Step at the Membrane

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP•magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP•SR targeting complexes

A luminescent oxygen channeling biosensor that measures small GTPase activation

We established a homogeneous luminescent oxygen channeling sensor for measuring activation states of small GTPases. The assay quantifies activated GTPases in cell lysates, can be applied to different GTPases, and can be used for multiplex screening. The study will provide guidelines for determining activation states of diverse GTPases in various biological contexts

The bacterial SRP receptor, SecA and the ribosome use overlapping binding sites on the SecY translocon

Signal recognition particle (SRP)-dependent protein targeting is a universally conserved process that delivers proteins to the bacterial cytoplasmic membrane or to the endoplasmic reticulum membrane in eukaryotes. Crucial during targeting is the transfer of the ribosome-nascent chain complex (RNC) from SRP to the Sec translocon. In eukaryotes, this step is co-ordinated by the SRbeta subunit of the SRP receptor (SR), which probably senses a vacant translocon by direct interaction with the translocon. Bacteria lack the SRbeta subunit and how they co-ordinate RNC transfer is unknown. By site-directed cross-linking and fluorescence resonance energy transfer (FRET) analyses, we show that FtsY, the bacterial SRalpha homologue, binds to the exposed C4/C5 loops of SecY, the central component of the bacterial Sec translocon. The same loops serve also as binding sites for SecA and the ribosome. The FtsY-SecY interaction involves at least the A domain of FtsY, which attributes an important function to this so far ill-defined domain. Binding of FtsY to SecY residues, which are also used by SecA and the ribosome, probably allows FtsY to sense an available translocon and to align the incoming SRP-RNC with the protein conducting channel. Thus, the Escherichia coli FtsY encompasses the functions of both the eukaryotic SRalpha and SRbeta subunits in one single protein

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle

The YidC insertase also integrates multispanning membrane proteins that had been considered to be exclusively SecYEG dependent. Only membrane proteins that require SecA can be inserted only via SecYEG. Targeting to YidC is SRP dependent, and the C-terminus of YidC cross-links to SRP, FtsY, and ribosomal subunits