Structural insights into tail-anchored protein binding and membrane insertion by Get3

摘要: Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor TA proteins. consists an α-helical subdomain enriched methionine glycine residues. We present structural biochemical analyses alone well complex with protein, ribosome-associated 4 (Ramp4). domains form extensive dimer interface that encloses 2 nucleotides head-to-head orientation zinc ion. Amide proton exchange mass spectrometry shows displays considerable flexibility solution maps protein-binding site to subdomain. non-hydrolyzable ATP analogue AMPPNP-Mg2+- ADP-Mg2+-bound crystal structures representing pre- posthydrolysis states both closed form. In absence cargo, hydrolysis does not seem be possible. Comparison ADP·AlF4−-bound structure transition state (Mateja A, et al. (2009) Nature 461:361–366) indicates how presence is communicated ATP-binding site. vitro insertion studies show recombinant inserts Ramp4 nucleotide- receptor-dependent manner. Although required for per se, it seems efficient insertion. postulate needed release from its receptor. Taken together, our results provide mechanistic insights into posttranslational by Get3.