Integration of transmembrane domains is regulated by their downstream sequences

Abstract

The Sec61 translocon catalyzes translocation of proteins into the endoplasmic reticulum and the lateral integration of transmembrane segments into the lipid bilayer. Integration is mediated by the hydrophobicity of a polypeptide segment consistent with thermodynamic equilibration between the translocon and the lipid membrane. Integration efficiency of a generic series of increasingly hydrophobic sequences (H-segments) was found to diverge significantly in different reporter constructs as a function of the ∼100 residues that are C-terminal to the H-segments. The hydrophobicity threshold of integration was considerably lowered through insertion of generic ∼20-residue peptides either made of flexible glycine-serine repeats, containing multiple negative charges, or consisting of an oligoproline stretch. A highly flexible, 100-residue glycine-serine stretch maximally enhanced this effect. The apparent free energy of integration was found to be changed by more than 3 kcal/mol with the downstream sequences tested. The C-terminal sequences could also be shown to affect integration of natural mildly hydrophobic sequences. The results suggest that the conformation of the nascent polypeptide in the protected cavity between the ribosome and translocon considerably influences the release of the H-segment into the bilayer.

Membrane Protein Integration and Topogenesis at the ER

“…The membrane acts as an entropic trap for the TM segment and chaperone binding to the lumenally exposed sequence as a ratchet. Consistent with this view, the conformational properties of a sequence up to 100 residues downstream of the Hsegment affected the hydrophobicity threshold of integration [52]. Integration was facilitated, when the sequence was flexible and extended, and inhibited when compact, reflecting the gain in entropy permitted by the downstream sequence.…”

An Update on Sec61 Channel Functions, Mechanisms, and Related Diseases

“…However, our own unpublished work suggests that special features downstream of the signal peptides can also play a distinct role, which may be particularly relevant in cotranslational translocation when a considerable stretch of a nascent precursor polypeptide accumulates at the interface between ribosome and Sec61 complex, i.e., prior to Sec61 channel opening (see above) and in posttranslational translocation (S. Haßdenteufel, personal communication). This is reminiscent of the effects of downstream sequences in the integration of transmembrane helices into the membrane (Junne and Spiess, 2017 ). Interestingly, yeast Sec62 and mammalian TRAP were found to affect the topology of transmembrane helices that do not promote a specific initial orientation of membrane protein precursors in the membrane (Reithinger et al, 2013 ; Sommer et al, 2013 ).…”

ER import of small human presecretory proteins: components and mechanisms

“…In any case, signal peptides start sampling the cytosolic funnel of the Sec61‐channel pore, that is start their dwell time in the Sec61‐channel pore as brilliantly visualized for cotranslational transport by Zhang and Miller (https://www.cell.com/cms/10.1016/j.celrep.2012.08.039/attachment/cd0b8007-ca63-44e5-afa1-454133428f79/mmc2.mp4). According to these simulations, sampling or dwell time is influenced by deleterious charges, hydrophobicity, mature protein domain length, and translation speed, which is dependent on pause sites, rare codons or hairpins in the mRNA and arrest peptides or polyproline motifs in the polypeptide . Nascent membrane proteins leave the Sec61‐channel with their transmembrane domain(s) (TMD) moving laterally into the phospholipid bilayer via the so‐called lateral gate, while the nascent precursors of soluble proteins leave it axially into the ER lumen.…”