Measuring Endoplasmic Reticulum Signal Sequences Translocation Efficiency Using the Xbp1 Arrest Peptide

Abstract

SummarySecretory proteins translocate across the mammalian ER membrane co-translationally via the ribosome-sec61 translocation channel complex. Signal sequences within the polypeptide, which guide this event, are diverse in their hydrophobicity, charge, length, and amino acid composition. Despite the known sequence diversity in the ER-targeting signals, it is generally assumed that they have a dominant role in determining co-translational targeting and translocation initiation process. We have analyzed co-translational events experienced by secretory proteins carrying efficient, versus inefficient (poorly hydrophobic) signal sequences, using an assay based on Xbp1 peptide-mediated translational arrest. With this method we were able to measure the functional efficiency of ER signal sequences. We show that an efficient signal sequence experiences a two-phases event in which the nascent chain is pulled from the ribosome during its translocation, thus resuming translation and yielding full-length products.Conversely, the inefficient signal sequence experiences a single weaker pulling event, suggesting inadequate engagement by the translocation machinery of these marginally hydrophobic signal sequences.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Preprotein signature for full susceptibility to the co‐translational translocation inhibitor cyclotriazadisulfonamide

“…Several research groups have proposed the “head‐in‐first” model, with the N‐terminal SP being initially inserted toward the ER lumen . In our previous report, we demonstrated that stalled nascent chains of huCD4 initially insert in an N lum /C cyt orientation before the final topology inversion of the SP takes place, in accordance with the “head‐in‐first” model.…”

“…During co‐translational translocation through the Sec61 translocon of uncleaved signal sequences (termed signal anchors), charges in the signal sequence were also shown to affect topology, similar to the “positive inside rule” . In addition, several studies propose the “head‐in‐first” model, with the N‐terminal SP being inserted towards the ER lumen and subsequently inverted to form a loop in the translocon, ready for cleavage by the signal peptidase on the luminal side of the ER, a model which we also proposed for huCD4 . The position of the lysine residues in huCD4 would favor an initial head‐in‐first insertion of the SP into the translocon channel, with the amino terminus facing the ER lumen and the C‐terminus at the cytoplasmic side of the ER membrane (N lum /C cyt ).…”

“…The first (weaker) pulling force exerted on the SP is during early (head‐in‐first) engagement of the nascent chain with the translocon channel, whereas the second (stronger) pulling phase represents the SP inversion inside the translocon. Interestingly, the force profile was highly dependent on the hydrophobicity of the h‐domain of the SP and on the positive charge of the N‐region of the SP . As we hypothesize that CADA stabilizes an intermediate folded state of the SP during the topology inversion, the compound/nascent chain/translocon complex should be able to resist that second pulling force to prevent further protein translocation.…”

“…The primary sequence of each SP will thus dictate the final functionality of the SP and subsequently determine the efficiency and speed of the “entry” of the substrate into the secretory pathway. A recent study with the Xbp1 arrest peptide by Kriegler et al . demonstrated that efficient (or “strong”) SPs experience a two‐phase pulling event whereby the nascent chain is pulled from the ribosome during its translocation.…”

Membrane translocation at the ER: with a little help from my friends

“…Once bound, SRP targets the ribosome-nascent chain (RNC) complex to the ER membrane by binding its cognate ER membrane-localized SRP receptor (SR) in a GTP-dependent manner [3], at which point the RNC is transferred to the Sec61 complex and the signal peptide engages its core Sec61a subunit in a 'head-on' orientation (ii). Strongly hydrophobic signal peptides are reoriented in the Sec61 complex [14], and this process likely initiates the opening of both the lateral gate, enabling access to the lipid bilayer, and the plug domain, enabling access to the ER lumen, that is the gating of the ER translocation channel (iii). In the case of secretory proteins, the ER signal peptide (orange) remains associated with the Sec61 complex until its cleavage by the signal peptidase complex (SPC) (iv).…”