title: Structural insight into the membrane insertion of tail-anchored proteins by Get3.
abstract: 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.
authors.affiliation: 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.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamagata
authors.forename: Hisatoshi
authors.initials: H
authors.lastname: Mimura
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sato
authors.forename: Masami
authors.initials: M
authors.lastname: Yamashita
authors.forename: Azusa
authors.initials: A
authors.lastname: Yoshikawa
authors.forename: Shuya
authors.initials: S
authors.lastname: Fukai
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D020662:Guanine Nucleotide Exchange Factors; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C529048:Get3 protein, S cerevisiae; D015032:Zinc