In vitro Assays for Targeting and Insertion of Tail‐Anchored Proteins Into the ER Membrane

Abstract

Membrane proteins mediate numerous essential cellular functions. Due to the aggregation propensity of hydrophobic transmembrane domains in aqueous environments, the targeting and insertion of membrane proteins pose major challenges to cells. In the Guided Entry of Tail-anchored protein (GET) pathway, an essential class of newly synthesized tail-anchored proteins (TAs) are chaperoned and guided by multiple targeting factors to the endoplasmic reticulum (ER) membrane. Deciphering the molecular mechanism of this cellular process has benefitted from successful in vitro reconstitution of individual molecular events in the GET pathway with purified components. Here we describe recently developed protocols for in vitro reconstitution of functional complexes of TA substrates with their targeting factors, for monitoring the transfer of TAs between targeting factors, and for the insertion of TA into the microsomal membrane. These procedures are generally applicable to the interrogation of other post-translational membrane protein targeting pathways.

A trap mutant reveals the physiological client spectrum of TRC40

“…6A) despite a TMS hydrophobicity score that would have predicted an interaction with TRC40 during biogenesis. We cannot exclude that such a failure to be trapped may reflect a low amount of TA protein precursor present in the cell, although ZFPL1 has been shown to readily detected in HeLa and HEK293 cells (Cho et al, 2018; Geiger et al, 2012). Generally, we found no indication that copy numbers or half-life – as available from systematic studies on protein turnover in HeLa cells (Cambridge et al, 2011; Cho et al, 2018) – correlated with the propensity of a TA protein to be trapped by TRC40 D74E .…”

“…We cannot exclude that such a failure to be trapped may reflect a low amount of TA protein precursor present in the cell, although ZFPL1 has been shown to readily detected in HeLa and HEK293 cells (Cho et al, 2018; Geiger et al, 2012). Generally, we found no indication that copy numbers or half-life – as available from systematic studies on protein turnover in HeLa cells (Cambridge et al, 2011; Cho et al, 2018) – correlated with the propensity of a TA protein to be trapped by TRC40 D74E . For example, TA proteins identified by SWATH mass spectrometry with high confidence ranged from 140 million molecules per cell and a half-life of 47 h (EMD) to 3 million and 20 h (Stx5) or even only 800,000 molecules per cell (UBE2J1).…”

“…For example, TA proteins identified by SWATH mass spectrometry with high confidence ranged from 140 million molecules per cell and a half-life of 47 h (EMD) to 3 million and 20 h (Stx5) or even only 800,000 molecules per cell (UBE2J1). In fact, based on the available data (Cambridge et al, 2011; Cho et al, 2018) VAPB (80 million molecules per cell and 42 h) and FIS1 (56 million and 40 h), and GOSR2 (10 million, 29 h) and PTP1B (6 million, 31 h) are pairs of TA proteins of very similar abundance and half-life that perfectly underscore TMS hydrophobicity as the dominant parameter as a prediction for TRC40 recruitment in live cells.…”

Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad

“…We first compared Sgt2-WT and Sgt2ΔN in a TA capture assay in which the TA substrate Bos1 was generated by in vitro translation (IVT) in the E. coli S30 extract devoid of GET pathway components (Fig. 2a ) 43 , 44 . An unnatural amino acid photocrosslinker, p-benzoylphenylalanine (Bpa), replaced Ala228 at the 6th residue in the Bos1-TMD during IVT 18 , 45 , 46 .…”

Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins

“…STX5 – a target-SNARE – is required for fusion of retrograde vesicles with the TGN, and knockdown of its expression is sufficient to trap ricin in the early endosome (Amessou et al, 2007; Dascher and Balch, 1996; Suga et al, 2005; Norlin et al, 2016; Bennett et al, 1993). Most SNAREs like STX5 are tail-anchored (TA) proteins – a class of membrane proteins with a single, C-terminal transmembrane domain (TMD) that are post-translationally targeted to the ER for insertion by the transmembrane domain recognition complex (TRC) pathway (Hegde and Keenan, 2011; Denic, 2012; Cho et al, 2018). Notably, our previous genome-wide CRISPR/Cas9 deletions screens have identified STX5 and its ER targeting factor, ASNA1 (also known as TRC40) among the top hits that confer resistance to ricin (Morgens et al, 2017).…”