Abstract
Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find that several newly synthesized tail-anchored proteins are transiently associated with a defined subset of cellular components. Among these, we identify several ER proteins, including subunits of the Sec61 translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal peptidase complex. When we analyze the cotranslational membrane insertion of a comparable signal-anchored protein we find the nascent polypeptide associated with a similar set of ER components. We conclude that the pathways for the integration of tail-anchored and signal-anchored membrane proteins at the ER exhibit a substantial degree of overlap, and we propose that this reflects similarities between co- and post-translational membrane insertion.
Highlights
Membrane protein insertion at the mammalian endoplasmic reticulum (ER) occurs most commonly via the cotranslational pathway, in which a hydrophobic signal sequence emerges from the ribosome and is recognized by the signal recognition particle (SRP)1 [1]
Discrete Cross-linking Products Are Observed with Newly Synthesized Sec61—We chose to investigate the association of newly synthesized tail-anchored proteins with ER components using the bifunctional cross-linking reagent BMH, which is highly specific for cysteine residues
During this study we have identified a discrete subset of cellular components that are transiently associated with newly membrane-inserted tail-anchored proteins
Summary
Antibodies—Polyclonal antibodies were raised against specific peptides representing Sec61␣, Sec61 The coding region was amplified by PCR, introducing a BglII site 1 base 5Ј to the initiation codon using primer 5Ј-ACTTTGGCAGATCTACCATGAAGGACCGAACCCAGG-3Ј and an XbaI site after the termination codon using primer 5Ј-CACCATCGGGGGCATCTTTGGATAGTCTAGATATA-3Ј This PCR product was cut with BglII and ligated into the BglII and HpaI sites of pSPUTK (Stratagene). A time course was performed to establish the amount of Sec61 that was integrated into canine pancreatic microsomes under the experimental conditions that we were using This analysis was carried out across a 60-min period, and the percentage integration was defined as the proportion of the total protein synthesized at a particular time point which was found to be membraneassociated and resistant to extraction with alkaline sodium carbonate solution [23, 32]. The resulting beads were heated to 70 °C for 10 min in SDS-PAGE sample buffer, and unless otherwise stated, the solubilized material was resolved on 16% polyacrylamide Tris-glycine gels run under denaturing conditions
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call