Abstract
The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.
Highlights
The Sec[61] complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway
After synthesis at the endoplasmic reticulum (ER), many soluble proteins including cytokines, hormones and enzymes follow the secretory pathway to the cell surface, where they are released by exocytosis[1]
To explore the generality of this inhibitory effect, we studied several human secretory proteins that are expressed in HepG2 cells using the same approach (Fig. 1a)
Summary
The Sec[61] complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. As nascent preproteins are translocated across the ER, SP cleavage occurs at the lumenal side of the membrane, together with modifications including N-glycosylation and disulphide bond formation[15,16] These events ensure the authentic conformational maturation of fully translocated soluble proteins that may remain ER residents or continue through the secretory pathway. Continued access to the ER lumen in the presence of the compound does not guarantee protein secretion and our data suggest that Ipom-F treatment leads to a defect in ER lumenal protein folding This defect most likely reflects an inability to replenish ER lumenal chaperones that results from two distinct consequences of Ipom-F treatment: firstly, prolonged exposure to Ipom-F results in an atypical ER stress response that fails to effectively upregulate the transcription of mRNAs encoding ER lumenal chaperones; secondly, the Sec61-dependent translocation of several abundant ER chaperones is strongly inhibited by Ipom-F. On the basis of these data, we conclude that Ipom-F directly inhibits protein translocation across the ER membrane and indirectly affects the protein folding capacity of the ER lumen, thereby providing a molecular basis for the atypical UPR that we find it to activate
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