Structural insight into the membrane insertion of tail‐anchored proteins by Get3

Tail anchored (TA) proteins, which are important for numerous cellular processes, are defined by a single transmembrane domain (TMD) near the C‐terminus. The membrane insertion of TA proteins is mediated by the highly conserved ATPase Get3. Here we report the crystal structures of Get3 in ADP‐bound and nucleotide‐free forms at 3.0 Å and 2.8 Å resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn2+‐mediated homodimer in a head‐to‐head orientation, representing an open dimer conformation. Our cross‐link experiments indicated the closed dimer‐stimulating ATP hydrolysis, which might be coupled with TA‐protein release. Further, our coexpression‐based binding assays using a model TA protein Sec22p revealed the direct interaction between the helical domain of Get3 and the Sec22p TMD. This interaction is independent of ATP and dimer formation. Finally, we propose a structural mechanism that links ATP hydrolysis with the TA‐protein insertion mediated by the conserved DTAPTGH motif.

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