Abstract
The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.
Highlights
The heterotrimeric Sec[61] complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP)
Following the resolution of radiolabelled products by SDSPAGE, we found that all five-model type III TMPs displayed apparently normal levels of N-glycosylation in the presence of 1 μM ipomoeassin F (Ipom-F) (Fig. 1d, lane 1 versus lane 3 in each panel)
Ipom-F consistently inhibits Sec61-mediated translocation in a substrate selective manner that emulates the actions of mycolactone[1,2,27] and suggests that the integration of type III TMPs is mechanistically distinct from other classes of single-pass membrane proteins
Summary
The heterotrimeric Sec[61] complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). 1234567890():,; Ipomoeassin F (Ipom-F)[1] and mycolactone[2,3] are natural products that inhibit the Sec61-mediated translocation of newly synthesised polypeptides into and across the endoplasmic reticulum (ER) membrane Both compounds are substrate selective; effectively blocking the translocation of secretory proteins and the ER integration of type I and type II transmembrane proteins (TMPs) (Fig. 1a), but not affecting two type III TMPs (glycophorin C; GypC and synaptotagmin 1; Syt1) studied to date[1,2,3]. It appears that the EMC and Sec[61] complex act together to provide a co-ordinated site for the membrane integration of a subset of multi-pass TMPs at the ER14,17,19–21
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