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Structure of the Sgt2/Get5 complex provides insights into GET-mediated targeting of tail-anchored membrane proteins

DOI: 10.1073/pnas.1207518110 DOI Help
PMID: 23297211 PMID Help

Authors: Aline C. Simon (University of Cambridge) , Peter J. Simpson (Imperial College London) , Rachael M. Goldstone (Imperial College London) , Ewelina M. Krysztofinska (Imperial College London) , James W. Murray (Institute for Cell and Molecular Biosciences, Newcastle University) , Stephen High (University of Manchester) , Rivka L. Isaacson (Imperial College London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences

State: Published (Approved)
Published: January 2013

Abstract: Small, glutamine-rich, tetratricopeptide repeat protein 2 (Sgt2) is the first known port of call for many newly synthesized tail-anchored (TA) proteins released from the ribosome and destined for the GET (Guided Entry of TA proteins) pathway. This leads them to the residential membrane of the endoplasmic reticulum via an alternative to the cotranslational, signal recognition particle-dependent mechanism that their topology denies them. In yeast, the first stage of the GET pathway involves Sgt2 passing TA proteins on to the Get4/Get5 complex through a direct interaction between the N-terminal (NT) domain of Sgt2 and the ubiquitin-like (UBL) domain of Get5. Here we characterize this interaction at a molecular level by solving both a solution structure of Sgt2_NT, which adopts a unique helical fold, and a crystal structure of the Get5_UBL. Furthermore, using reciprocal chemical shift perturbation data and experimental restraints, we solve a structure of the Sgt2_NT/Get5_UBL complex, validate it via site-directed mutagenesis, and empirically determine its stoichiometry using relaxation experiments and isothermal titration calorimetry. Taken together, these data provide detailed structural information about the interaction between two key players in the coordinated delivery of TA protein substrates into the GET pathway.

Journal Keywords: Biophysical; Carrier; Crystallography; X-Ray; Humans; Membrane; Metabolic; Models; Molecular; Multiprotein; Mutagenesis; Site-Directed; Nuclear; Biomolecular; Protein; Recombinant; Saccharomyces; Sequence; Amino; Ubiquitin; Ubiquitins

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography

Added On: 14/01/2013 15:37

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)