α-Helical Domains Promote Translocation of Intrinsically Disordered Polypeptides into the Endoplasmic Reticulum

Abstract

Co-translational import into the endoplasmic reticulum (ER) is primarily controlled by N-terminal signal sequences that mediate targeting of the ribosome-nascent chain complex to the Sec61/translocon and initiate the translocation process. Here we show that after targeting to the translocon the secondary structure of the nascent polypeptide chain can significantly modulate translocation efficiency. ER-targeted polypeptides dominated by unstructured domains failed to efficiently translocate into the ER lumen and were subjected to proteasomal degradation via a co-translocational/preemptive pathway. Productive ER import could be reinstated by increasing the amount of ␣-helical domains, whereas more effective ER signal sequences had only a minor effect on ER import efficiency of unstructured polypeptides. ER stress and overexpression of p58 IPK promoted the co-translocational degradation pathway. Moreover polypeptides with unstructured domains at their N terminus were specifically targeted to proteasomal degradation under these conditions. Our study indicates that extended unstructured domains are signals to dispose ER-targeted proteins via a co-translocational, preemptive quality control pathway.

The α-Helical Structure of Prodomains Promotes Translocation of Intrinsically Disordered Neuropeptide Hormones into the Endoplasmic Reticulum

“…Yellow, small ribosomal subunit; gray, large ribosomal subunit; green, tRNA; blue, translocon. The model is based on the experiments described in this study and previous work (19,57).…”

“…Secretome-Previous studies on the biogenesis of pathogenic mutants of the prion protein and model substrates revealed that IDPs equipped with an N-terminal signal peptide are inefficiently imported into the ER (18,19,44). Based on these findings, we performed an in silico screen for the identification of secretory proteins that are dominated by intrinsically disordered domains (Ͼ70% of the amino acid sequence).…”

“…Heterologous ␣-Helical Domains Restore Secretion of Mutants Devoid of the Prodomain-It was shown previously that impaired ER import of IDDs could be restored by ␣-helical domains (18,19). To address the possibility that the activity to promote secretion of the hormone domains does not reside in the amino acid sequence of the prodomains but rather in their ability to adopt an ␣-helical conformation, we performed domain-swapping experiments.…”

“…In previous work using pathogenic prion protein mutants and model substrates, we identified an additional element to regulate translocation efficiency: the secondary structure of the nascent chain (18,19). Specifically, we found that intrinsically disordered proteins (IDPs) equipped with an N-terminal ER signal peptide are not efficiently translocated into the ER.…”

“…Specifically, we found that intrinsically disordered proteins (IDPs) equipped with an N-terminal ER signal peptide are not efficiently translocated into the ER. ER import of these proteins could be restored by fusing ␣-helical domains to the IDPs (19). In this context, it is important to note that the nascent chain can adopt a stable secondary structure already in the ribosomal exit tunnel (20 -25).…”