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

Structural Biology Laboratory, Life Science Division, Synchrotron Radiation Research Organization and Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.
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 A and 2.8 A resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn(2+)-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.
Mesh Terms:
Adenosine Triphosphatases, Adenosine Triphosphate, Binding Sites, Cell Membrane, Cross-Linking Reagents, Crystallography, X-Ray, Guanine Nucleotide Exchange Factors, Hydrolysis, Models, Biological, Mutation, Protein Binding, Protein Multimerization, Protein Structure, Secondary, Protein Structure, Tertiary, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Zinc
Genes Cells Jan. 01, 2010; 15(1);29-41 [PUBMED:20015340]
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