Hydrophobicity, rather than secondary structure, is essential for the SRP dependent targeting of GPR35 to the ER membrane

Jon K Cherry,Cheryl A Woolhead

doi:10.1007/s10863-019-9785-0

Jon K Cherry, Cheryl A Woolhead

Open Access

https://doi.org/10.1007/s10863-019-9785-0

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Abstract

The folding and targeting of hydrophobic transmembrane domains poses a major challenge to the cell. Several membrane proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. However, there is little information on one of the largest classes of eukaryotic membrane proteins; the G protein-coupled receptors (GPCRs). In this study we show that the signal anchor domain of GPR35 remains in an extended conformation whilst exiting the ribosome tunnel, the polypeptide chain then forms interactions with components of the SRP targeting pathway, and the Sec61 translocon, resulting in a compacted conformation prior to integration into the ER membrane. We conclude that transmembrane structure is most likely adopted after the domain leaves the ribosome tunnel and that the interaction of the signal anchor with SRP is dependent on the native levels of hydrophobicity within the first transmembrane domain. Therefore, we propose a mechanism by which the first transmembrane domains of multi-spanning membrane proteins adopt compacted structures following SRP targeting but before insertion into the ER membrane.

Highlights

The co-translational targeting of integral membrane proteins (IMPs) poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer, via the Se61 machinery (SecYEG in bacteria)
Conditions within both the prokaryotic and eukaryotic tunnel have proven themselves favourable for generation of structured nascent peptides such as α-helices and tertiary hairpins (Thommen et al 2017). To determine whether such structural formation is likely to occur in the signal anchor domain of a model G protein-coupled receptors (GPCRs); GPR35, a study was set up to investigate where, if at all, the first TM domain is likely to form a structured polypeptide
To explore the formation of secondary structure within the signal anchor domain of GPR35, ribosome nascent chain complexes (RNCs) were synthesized in vitro, with individual intermediates generated from linear DNA lacking a stop codon

Summary

Introduction

The co-translational targeting of integral membrane proteins (IMPs) poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer, via the Se61 machinery (SecYEG in bacteria). The wall of the tunnel is lined with ribosomal RNA and ribosomal proteins (uL4, uL22 and uL23), which have been identified to interact with the nascent chain in a sensory and regulatory manner, often influencing processes such as biogenesis, targeting and membrane insertion. Several studies in the last decade have provided evidence of ribosomal proteins line, but protrude into the tunnel, generating distinct zones of helix stabilization that could play a key role in promoting peptide folding (Bhushan et al 2010; Lu and Deutsch 2005b, 2008; Woolhead et al 2004)

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