The structure of a tail-anchor membrane protein-binding complex reveals the regulation of Get3 by Get4

Abstract

Tail-anchored (TA) membrane proteins are targeted to the endoplasmic reticulum via the post-translational GET pathway. A critical step in this essential pathway is the transfer of TA substrates from the co-chaperone Sgt2 to the Get3 ATPase, mediated by the hetero-tetrameric complex Get4/Get5 (Get4/5). However, a major outstanding question is how Get4 binds to Get3 and regulates its activity. Here we report a 5.4 Å crystal structure of an ATP-bound Get3-Get4/5 complex from Saccharomyces cerevisiae (Sc), the largest pathway components solved to date. Our structure reveals the role of nucleotide in complex formation and the presence of two functionally distinct binding interfaces. Mutational analysis confirms that one interface is required for the specificity of binding, while the other interface is involved in the regulation of Get3 ATPase activity. Additional functional assays demonstrate that Get4/5-mediated regulation of ATP hydrolysis by Get3 is essential for efficient TA protein targeting. Our work also supports a model for Get3/TA complex formation in which two opposing Get3 dimers are bridged by a single Get4/5 hetero-tetramer. Finally, we present a 2.8 Å crystal structure of an apo ScGet3-Get4/5 complex that provides evidence for a rapid initial binding state mediated by electrostatics. This work illustrates how Get4/5 regulates Get3, priming it for TA loading, a critical step in this important pathway.

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