A protean clamp guides membrane targeting of tail-anchored proteins

Abstract

Proper localization of proteins to target membranes is a fundamental cellular process. How the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. Here, we address this question for the conserved ATPase guided entry of tail-anchored protein 3 (Get3), which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER). Single-molecule fluorescence spectroscopy showed that, contrary to previous models of a static closed Get3•TA complex, Get3 samples open conformations on the submillisecond timescale upon TA binding, generating a fluctuating "protean clamp" that stably traps the substrate. Point mutations at the ATPase site bias Get3 toward closed conformations, uncouple TA binding from induced Get3•Get4/5 disassembly, and inhibit the ER targeting of the Get3•TA complex. These results demonstrate an essential role of substrate-induced Get3 dynamics in driving TA targeting to the membrane, and reveal a tightly coupled channel of communication between the TA-binding site, ATPase site, and effector interaction surfaces of Get3. Our results provide a precedent for large-scale dynamics in a substrate-bound chaperone, which provides an effective mechanism to retain substrate proteins with high affinity while also generating functional switches to drive vectorial cellular processes.protein targeting | chaperones | protein dynamics | single-molecule spectroscopy | ATPases O ver 35% of proteins need to be localized to the correct cellular destinations after their initial synthesis in the cytosol. These protein-targeting processes are essential for the establishment and maintenance of compartmentalization in all cells and pose complex mechanistic challenges to targeting machineries. To minimize improper exposure of substrate proteins in the cytosol, targeting factors must bind substrate proteins with high stability. This requirement is especially stringent during the targeting of integral membrane proteins, whose high aggregation propensity in the cytosol and other aqueous cellular environments demands that targeting factors also serve as effective chaperones to protect substrates from aggregation. Further, to minimize futile cycling of targeting factors, loading of substrates on the targeting factor must be tightly coupled to their delivery to membrane receptor sites. Finally, once at the target membrane, the targeting machinery must readily switch to a lowaffinity state to release substrate proteins to receptor complexes, translocases, or the phospholipid bilayer. With a few exceptions (1, 2), the nature and dynamics of protein targeting complexes and how their biophysical properties help meet these complex functional demands are not well understood, especially for posttranslational protein targeting pathways.

Guiding tail-anchored membrane proteins to the endoplasmic reticulum in a chaperone cascade

“…In this work, the open-toclosed conformational rearrangements of Get3 were directly monitored using a pair of FRET dyes incorporated at its helical domains (Fig. 4B, green and red stars) (54). Contrary to the accepted models, this study found that the TA substrate initiates sub-millisecond time-scale opening motions in Get3 that drive the targeting phase of the pathway (Fig.…”

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

“…Inclusion of Sgt2 and Get4/5 is optional and does not substantially affect the activity or conformation of Get3•TA (Chio, Chung, Weiss, & Shan, 2017), but lengthens the subsequent purification of the Get3•TA due to Get4/5 association with Get3. The efficiency of TA targeting and insertion from the purified Get3•TA into yeast rough ER microsomes (yRM) is assessed via glycosylation of an engineered opsin tag at the C-terminus of TA substrates (Fig.…”

“…Co-expression of model TAs with SGTA (the mammalian homolog of Sgt2) or with Get3 in E. coli provides a facile method to generate recombinant SGTA•TA or Get3•TA complexes (Mateja et al, 2015; Mock et al, 2015; Rome, Chio, Rao, Gristick, & Shan, 2014; Rome, Rao, Clemons, & Shan, 2013). Nevertheless, the stoichiometry of the Get3•TA complex appears to be highly dependent on the level of protein expression (Chio, Chung, et al, 2017; Mateja et al, 2015; Suloway, Rome, & Clemons, 2012), raising questions as to whether the composition and activity of Get3•TA are perturbed by recombinant overexpression. The recombinant SGTA•TA complex also exhibited a low efficiency (5–10%) of TA transfer to Get3 (Mock et al, 2015).…”

“…In vitro translation based on the E. coli S30 extract supplemented with near physiological concentrations of Sgt2 or Get3 provides a facile alternative platform to reconstitute targeting complexes with high activity (Chio, Chung, et al, 2017; Rao et al, 2016). Coupling this platform to the amber suppression system (J.…”

Receptor compaction and GTPase movements drive cotranslational protein translocation

“…Analysis of the dynamics of the rearrangements supported this sequential model. We first performed burst variance analysis (BVA), which detects dynamics by comparing the standard deviation of E* (sE*) for individual molecules to the static limit, defined by photon statistics (Supplementary Methods) 27,[29][30][31] . If multiple conformations interconvert on the sub-millisecond timescale, the observed sE* would be higher than the static limit, whereas sE* would lie on the static limit curve if conformational interconversions are slower compared to molecular diffusion (1-5 milliseconds) 27,[29][30][31] .…”