In Vitro Dissection of Protein Translocation into the Mammalian Endoplasmic Reticulum

Abstract

In eukaryotic cells, roughly one-fourth of all mRNAs code for secretory and membrane proteins. This class of proteins must first be segregated to the endoplasmic reticulum, where they are either translocated into the lumen or inserted into the lipid bilayer. The study of these processes has long relied on their successful reconstitution in cell-free systems. The high manipulability of such in vitro systems has allowed the identification of key machinery, elucidation of their functional roles in translocation, and dissection of their mechanisms of action. Here, we provide the basic methodology for (i) setting up robust mammalian-based in vitro translation and translocation systems, (ii) assays for protein translocation, insertion, and topology, and (iii) methods to solubilize, fractionate, and reconstitute ER membranes. Variations of these methods should be applicable not only to forward protein translocation systems but also for dissecting other poorly understood membrane-associated processes such as retrotranslocation.

Structural basis for stop codon recognition in eukaryotes

“…The remaining C-terminal portion of the protein was deleted using Phusion mutagenesis (Thermo Scientific). In vitro transcription reactions were performed using PCR products generated with primers flanking the SP6 promoter and the 3’UTR of the SP64 vector as previously described 31 . The cDNA encoding human eRF1 (Origene) was subcloned into a pRSETA expression vector after an N-terminal 6× His tag and TEV cleavage site using standard procedures.…”

“…In vitro translations in a rabbit reticulocyte (RRL) system were for 25 min at 32°C as before 30,31 . Where indicated, 0.5 μM eRF1 AAQ was included to trap termination complexes.…”

Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes

“…To generate elongation complexes, the open reading frame of KRas was cloned after a 3X Flag tag in an SP64-based plasmid using conventional techniques. In vitro transcription reactions were performed using PCR products generated with primers that amplify from the SP6 promoter to either the 3′ UTR of the SP64 vector (Sharma et al., 2010) or to directly after Val68 of Sec61β (Shao et al., 2013). …”

“…Transcription reactions were conducted with ∼5-20 ng/μl purified PCR product, in 40 mM HEPES pH 7.4, 6 mM MgCl 2 , 20 mM spermidine (Sigma), 10 mM DTT, 0.5 mM ATP, 0.5 mM UTP, 0.5 mM CTP, 0.1 mM GTP (Roche), 0.5 mM CAP (NEB), 0.4-0.8 U/μL rRNasin (Promega), and 0.4 U/μL SP6 polymerase (NEB) at 37°C for 60 min (Sharma et al., 2010). In vitro translation reactions in a homemade rabbit reticulocyte (RRL) system containing 1/20 volume of transcription reaction, 0.5 μCi/μL 35 S-methionine (Perkin Elmer EasyTag), nuclease-treated crude rabbit reticulocyte (Green Hectares), 20 mM HEPES, 10 mM KOH, 40 μg/mL creatine kinase (Roche), 20 μg/mL pig liver tRNA, 12 mM creatine phosphate (Roche), 1 mM ATP (Roche), 1 mM GTP (Roche), 50 mM KOAc, 2 mM MgCl 2 , 1 mM glutathione, 0.3 mM spermidine, and 40 μM of each amino acid except for methionine (Sigma), were at 32°C for 25 min unless otherwise indicated (Shao et al., 2013, Sharma et al., 2010).…”

“…In vitro translation reactions in a homemade rabbit reticulocyte (RRL) system containing 1/20 volume of transcription reaction, 0.5 μCi/μL 35 S-methionine (Perkin Elmer EasyTag), nuclease-treated crude rabbit reticulocyte (Green Hectares), 20 mM HEPES, 10 mM KOH, 40 μg/mL creatine kinase (Roche), 20 μg/mL pig liver tRNA, 12 mM creatine phosphate (Roche), 1 mM ATP (Roche), 1 mM GTP (Roche), 50 mM KOAc, 2 mM MgCl 2 , 1 mM glutathione, 0.3 mM spermidine, and 40 μM of each amino acid except for methionine (Sigma), were at 32°C for 25 min unless otherwise indicated (Shao et al., 2013, Sharma et al., 2010). …”