A previously described native human Sec61β construct (7 (link)) was modified to contain the TMD from VAMP, followed by a C-terminal opsin tag (fig. S1 ). This ORF was subcloned in place of DHFR in the control T7-driven plasmid for in vitro transcription and translation in the PURE system (NEB). Yeast Get3 antibody was as described previously (10 (link)). The SGTA antibody was generated against a synthetic C-terminal peptide conjugated to KLH. The SGTA sequence used was CRSRRPSASNDDQQE, with the extra cysteine added at the N-terminus for KLH conjugation.
Chaperone-TA complexes were obtained by supplementing the PURE translation system (NEB) with plasmid encoding the VAMP TMD-containing substrate, 35S-methionine and 25 μM of purified Sgt2, SGTA or Get3. After incubating for 90 min at 37 °C, reactions were diluted with ice cold assay buffer (50 mM Hepes pH 7.4, 125 mM KOAc, 2 mM MgCl2), and separated at 4 °C through a 5-25% sucrose gradient (55,000 rpm/5 h in a TLS55 rotor); fractions containing the soluble complexes (seefig. S1 and S3 ) were pooled and either used immediately or frozen in liquid nitrogen and stored at −80 °C.
Substrate transfer reactions were carried out in 50 mM Hepes pH 7.4, 125 mM KOAc, 4 mM MgCl2 and 1 mM ATP and subjected to amine-reactive, sulfhydryl-reactive, or UV crosslinking. Amine-reactive crosslinking used 250 μM disuccinimidyl suberate (Pierce) at 22° C for 30 min. Sulfhydryl-reactive crosslinking used 200 μM bismaleimidohexane (Pierce) for 30 min on ice. Photo-crosslinking via BpF with UV was for 15 min on ice using a 365 nm longwave UV spot lamp (UVP) placed 10 cm from the sample. All crosslinking reactions were terminated by addition of excess SDS-PAGE buffer, followed optionally by immunoprecipitation (with anti-SGTA or anti-Get3 antibodies), separation by SDS-PAGE on 12% Tris-Tricine gels, Coomassie blue staining and autoradiography.
Size analysis of the Get3-TA substrate complexes formed by Get4-Get5-dependent loading from SGTA was performed using high-resolution 5-25% sucrose gradients (55,000 rpm/5 h in a TLS55 rotor); gradient fractions were analyzed by SDS-PAGE and quantified by phosphorimaging. Free, dimeric Get3 and E. coli-produced tetrameric Get3-TA substrate complexes (described above) were used as molecular weight standards; samples were analyzed by Coomassie staining and quantified by densitometry.
Insertion activity was analyzed by incubating TA substrate complexes (as indicated in the Figure legends) with yeast rough microsomes, prepared from wild-type yeast essentially as described previously (14 (link)). Insertion was monitored by TA protein glycosylation and quantified by phosphorimaging.
Chaperone-TA complexes were obtained by supplementing the PURE translation system (NEB) with plasmid encoding the VAMP TMD-containing substrate, 35S-methionine and 25 μM of purified Sgt2, SGTA or Get3. After incubating for 90 min at 37 °C, reactions were diluted with ice cold assay buffer (50 mM Hepes pH 7.4, 125 mM KOAc, 2 mM MgCl2), and separated at 4 °C through a 5-25% sucrose gradient (55,000 rpm/5 h in a TLS55 rotor); fractions containing the soluble complexes (see
Substrate transfer reactions were carried out in 50 mM Hepes pH 7.4, 125 mM KOAc, 4 mM MgCl2 and 1 mM ATP and subjected to amine-reactive, sulfhydryl-reactive, or UV crosslinking. Amine-reactive crosslinking used 250 μM disuccinimidyl suberate (Pierce) at 22° C for 30 min. Sulfhydryl-reactive crosslinking used 200 μM bismaleimidohexane (Pierce) for 30 min on ice. Photo-crosslinking via BpF with UV was for 15 min on ice using a 365 nm longwave UV spot lamp (UVP) placed 10 cm from the sample. All crosslinking reactions were terminated by addition of excess SDS-PAGE buffer, followed optionally by immunoprecipitation (with anti-SGTA or anti-Get3 antibodies), separation by SDS-PAGE on 12% Tris-Tricine gels, Coomassie blue staining and autoradiography.
Size analysis of the Get3-TA substrate complexes formed by Get4-Get5-dependent loading from SGTA was performed using high-resolution 5-25% sucrose gradients (55,000 rpm/5 h in a TLS55 rotor); gradient fractions were analyzed by SDS-PAGE and quantified by phosphorimaging. Free, dimeric Get3 and E. coli-produced tetrameric Get3-TA substrate complexes (described above) were used as molecular weight standards; samples were analyzed by Coomassie staining and quantified by densitometry.
Insertion activity was analyzed by incubating TA substrate complexes (as indicated in the Figure legends) with yeast rough microsomes, prepared from wild-type yeast essentially as described previously (14 (link)). Insertion was monitored by TA protein glycosylation and quantified by phosphorimaging.
