Summary-Paper 27

Summary- paper 27: Mechanism of signal-anchor triage during early steps of membrane protein insertion

Haoxi Wu, Ramanujan S. Hegde

Molecular Cell, 2023

Questions/gaps addressed:

• Key step in the biogenesis of a membrane protein is the insertion of its TMDs into the lipid bilayer in the correct topology. How is this coordinated? Key players involved: Sec61 translocon, EMC insertase, SRP and SRP receptor.

• Factors thought to be important:
  • for about half of the membrane proteins, the first TMD serves as a signal sequence (signal anchor) with a preceding N-tail.
  • how is it decided whether the N tail is in the lumen or cytosol? has been atrributed to flanking positive charge in the cytosol, signal anchor length and hydrophobilicity
• After co-translational targeting of an signal anchor to the ER, those intended for an N(exo) topology are inserted by EMC whereas N(cyt) are inserted by Sec61 translocon. This paper interrogates this further.

Key methods:

• Model substrate used: N(exo) signal anchor from trace-amine-associated receptor 5 (TAAR5), a G-protein-coupled receptor inserted by EMC.

• In vitro translation of 35S-labeled TAAR5-SA in the presence of semi-permeabilized cells. Addition of a glycoslation site at the N terminus allowed monitoring glycosylation as a proxy for translocation. strongly EMC dependent ( blocked in EMC6 KO) and mostly Sec61 independent (no effect of Apratoxin A (Sec61 inhibitor)).

• Translation stalled at a defined site using rare codons, the ribosome-nascent-chain complexes (RNCs) purified by sucrose-gradient fractionation, followed by incubation with semi-permeabilized cells for 10 min. Translocation of stalled at Signal anchor +50 and 70 aa strongly EMC dependent and mostly Sec61 independent. Signal anchor +30aa was Sec61 dependent and EMC independent.

• Used cysteine-reactive chemical crosslinking with bismaleimidohexane (BMH): produced sucrose-gradient-purified 35S-labeled TAAR5-SA+70 RNCs containing a single cysteine in the N-tail and crosslinking with BMH.

Major takeaways:

• A two-step sequential triage by EMC and Sec61 mediates the insertion and topogenesis of signal anchors.

• Membrane protein topology is not determined only after RNC delivery to Sec61. Found an additional step between SRP-mediated targeting and ribosome docking at Sec61. EMC transiently samples signal anchors at an earlier step and inserts a subset of them in the N(exo) topology. Those signal anchors that are skipped by EMC arrive at Sec61, which preferentially favors SA insertion in the N(cyt) topology.