Molecular mechanism of co-translational protein targeting by the signal recognition particle

Abstract

The signal recognition particle (SRP) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper localization, and has often served as a paradigm for understanding the molecular basis of protein localization within the cell. The SRP pathway exemplifies several key molecular events required for protein targeting to cellular membranes: the specific recognition of signal sequences on cargo proteins, the efficient delivery of cargo to the target membrane, the productive unloading of cargo to the translocation machinery and the precise spatial and temporal coordination of these molecular events. Here we highlight recent advances in our understanding of the molecular mechanisms underlying this pathway, and discuss new questions raised by these findings.

Figure 1

Overview of the pathway and components of SRP-dependent protein targeting. (A) Multiple pathways deliver proteins to the ER or plasma membrane, with the SRP pathway mediating the co-translational targeting of translating ribosomes whereas the SecA/B and alternative pathways mediating the post-translational targeting of proteins. (B) Overall structure of the mammalian (upper) and bacterial (lower) SRP and binding sites for the SRP protein subunits. The Alu- and S-domains of SRP and Domains I–IV of the 7S SRP RNA are indicated. Adapted from reference (5). (C) Domain structure of the SRP54 (or Ffh) protein. (D) Domain organization of the eukaryotic (left) and bacterial (right) SRP receptor.

Figure 2

A structural model of the SRP54 M-domain bound to the signal sequence and the SRP RNA. The crystal structure of the SRP54 M-domain (blue) in complex with a signal peptide (red; PDB ID 3KL4) was superimposed onto the structure of the Ffh M-domain in complex with a fragment of the SRP RNA (magenta; PDB ID 1DUL).

Figure 3

Conformational changes in the SRP and SR GTPases ensure the efficiency and fidelity of protein targeting. The steps are numbered to be consistent between parts (A) and (B). T and D denote GTP and GDP, respectively, (A) A series of discrete rearrangements occur during the SRP-SR interaction and can be regulated by the cargo and membrane translocon. ⊥ denotes the effect of cargo in disfavoring the rearrangements to the closed and activated states. Top panel: the crystal structures of free Ffh (blue; 1JPJ) and FtsY (green; 1Q9B) NG-domains bound to GMPPNP (spacefill). The IBD loops in both proteins are highlighted in red. Right panel: molecular model of the early intermediate (63) with Ffh and FtsY in blue and green, respectively. Bottom panel: G-domain superposition of the co-crystal structure of the Ffh-FtsY NG domain complex (1RJ9; Ffh and FtsY in blue and green, respectively) with those of the free proteins (grey). Left panel: Co-crystal structure of the Ffh-FtsY NG domain complex (1RJ9) highlighting the IBD loops (red) and catalytic interactions in the GTPase active site (zoom-in), with the GMPPCP molecules from Ffh and FtsY in blue and green, respectively, active site Mg²⁺ in magenta, nucleophilic waters (W) in blue, and the side chains of catalytic residues in the IBD loops in red. (B) GTPase rearrangements provide multiple regulatory points during protein targeting. Step 1, a cargo with a signal sequence (magenta) enters the pathway upon binding SRP. Step 2, SRP associates with SR to form a targeting complex, which is stabilized by the cargo in the early conformation. Step 3, association of SR with phospholipids is proposed to drive rearrangement to the closed state, during which SRP weakens its affinity for the cargo. Step 4, interaction of SR with the translocon could further allow the SRP•SR complex to rearrange to the activated state, which drives the handover of cargo from the SRP to the translocon. Step 5, GTP hydrolysis drives the disassembly and recycling of SRP and SR. At each step, the cargo can be either retained in (black arrows) or rejected from (red arrows) the SRP pathway.