Abstract

Protein translocation across the endoplasmic reticulum (ER) membrane is fundamental for protein sorting and secretion in all kingdoms. Translocation occurs through a protein-conducting channel in the ER membrane, which is formed by Sec61. Targeting of ribosome-nascent chain complexes (RNCs) to Sec61 is mediated by the signal recognition particle (SRP) and its cognate receptor (SR). However, Sec61 has a high affinity for nontranslating ribosomes and it is largely occupied in vivo. We addressed how RNC-SRP complexes can efficiently associate with the rER membrane, although most Sec61 seems to be occupied. We found that the spontaneous dissociation of ribosomes from the ER membrane is extremely slow. Surprisingly, membrane binding of RNC-SRP complexes does not require or cause the dissociation of prebound ribosomes. Instead, RNC-SRP complexes use a Sec61 population for translocation that cannot be bound by nontranslating ribosomes or RNCs alone. Complex formation between RNC-SRP and SR at the ER membrane facilitates the interaction between the RNC and the free Sec61. We have used biochemical and structural approaches to investigate why the free Sec61 fails to be bound by nontranslating ribosomes. Our data suggests that Sec61 is present in two interconvertible forms in the membrane, that are in an equilibrium with each other and provide high-affinity and low-affinity binding sites for ribosomes. The former are quickly occupied, while the latter remain free. The high-affinity binding sites are formed by tetrameric rings of Sec61, which provide several connections that each can break and reform, but together prevent the ribosome from detachment. In contrast, the low-affinity binding sites may correspond to lower oligomeric states of Sec61. Consistent with that, we could capture monomers or dimers of Sec61 bound to eukaryotic ribosomes by electron cryo-microscopy. A ribosome-bound Sec61 dimer was only observed in presence of a nascent chain, which could indicate that Sec61 oligomerization is induced by translocation of a nascent chain. We obtained similar results for bacterial ribosome-channel complexes. However, the low-affinity binding site seems to be more abundant in bacterial membranes, which indicates that the prokaryotic equilibrium between low- and high-affinity binding sites is adjusted differently from eukaryotes. 1 We propose a model, in which Sec61 exists in different oligomeric states in the ER membrane. Tetrameric Sec61 provides a high-affinity binding site for ribosomes and is readily occupied in vivo. Monomeric or dimeric Sec61 is accessible for targeting by the SRP pathway, which facilitates the interaction between RNC and free Sec61. After transfer of the nascent chain into the channel and dissociation of SR and SRP, more Sec61 molecules could be recruited to stabilize the interaction with the translating ribosome. A dynamic oligomerization equilibrium of Sec61 is thus the core of a mechanism that would guarantee efficient protein translocation in vivo.