Wild-type and selenomethionyl proteins were expressed in E. coli and purified using Ni-NTA and size-exclusion chromatography. Crystals were grown in hanging drop, vapour diffusion format. Diffraction data were collected from cryo-protected crystals at beamline 21-IDG of the Advanced Photon Source (Argonne National Laboratories). The structure of S. cerevisiae Get3 complexed with ADP·AlF 4− was determined by single-wavelength anomalous dispersion (SAD) from selenomethionine-containing protein using PHENIX33 (link). The structures of nucleotide-free S. cerevisiae and S. pombe Get3 were solved by molecular replacement in PHASER34 (link). A monomer of the S. cerevisiae Get3–ADP·AlF 4− complex (with nucleotide and the α-helical subdomain removed) was used as the search model. Refinement and model building were carried out with PHENIX33 (link) and COOT35 (link).
A series of GET3 genes containing site-specific mutations were generated by Quik-Change mutagenesis. The identity of each mutant was confirmed by DNA sequencing. Proteins were expressed in E. coli and purified by Ni-NTA chromatography. TA substrate binding was monitored using a native pull-down assay in which full-length 35S-labelled human SEC61β was translated in a TRC40-depleted reticulocyte lysate translation extract with or without recombinant wild-type or mutant Get3 protein. After translation, Get3 was immunoprecipitated under native conditions, analysed by SDS–PAGE, and quantified by phosphorimaging. The ATPase activity of Ni-NTA-purified protein was determined using an NADH-coupled microplate photometric assay36 (link),37 (link)Wild-type and mutant GET3 genes were subcloned into a low copy number URA plasmid under the control of a medium-strength, constitutive ACT1 promoter, and transformed into a Δget3 strain (Open Biosystems); serial dilutions of each transformant were spotted (along with wild type and vector only controls) onto synthetic defined medium (−uracil) supplemented with 100 μg ml−1 hygromycin B. Plates were photographed after 2 days at 37 °C.
A series of GET3 genes containing site-specific mutations were generated by Quik-Change mutagenesis. The identity of each mutant was confirmed by DNA sequencing. Proteins were expressed in E. coli and purified by Ni-NTA chromatography. TA substrate binding was monitored using a native pull-down assay in which full-length 35S-labelled human SEC61β was translated in a TRC40-depleted reticulocyte lysate translation extract with or without recombinant wild-type or mutant Get3 protein. After translation, Get3 was immunoprecipitated under native conditions, analysed by SDS–PAGE, and quantified by phosphorimaging. The ATPase activity of Ni-NTA-purified protein was determined using an NADH-coupled microplate photometric assay36 (link),37 (link)Wild-type and mutant GET3 genes were subcloned into a low copy number URA plasmid under the control of a medium-strength, constitutive ACT1 promoter, and transformed into a Δget3 strain (Open Biosystems); serial dilutions of each transformant were spotted (along with wild type and vector only controls) onto synthetic defined medium (−uracil) supplemented with 100 μg ml−1 hygromycin B. Plates were photographed after 2 days at 37 °C.
