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. Author manuscript; available in PMC: 2012 Sep 1.
Published in final edited form as: Traffic. 2011 Jul 3;12(9):1119–1123. doi: 10.1111/j.1600-0854.2011.01229.x

Figure 3. TA substrate binding and insertion by MtTRC40.

Figure 3

(a) Full-length human Sec61β, an insertion-deficient triple arginine mutant (3R) or a construct lacking its TMD (ΔTMD) were synthesized in RRL in the presence or absence of 100 ng/μl recombinant 6xHis-tagged MtTRC40. Fractions from a sucrose gradient were treated with an amine-specific crosslinker and pulled-down using anti-TRC40 antibodies (‘TRC40-IP’) or Ni-NTA sepharose beads (‘Ni-pull’). The positions of non-crosslinked Sec61β and the major crosslinked partner (endogenous mammalian TRC40 or recombinant MtTRC40) are indicated. (b) Sec61β was synthesized in PD-RRL supplemented with 5 ng/μl MtTRC40, incubated with liposomes or yeast rough microsomes prepared from a ΔGet3 strain (ΔGet3-yRM), and then subjected to chemical crosslinking. (c) Sec61β was synthesized in PD-RRL supplemented with 5 ng/μl MtTRC40. After incubation with with the indicated vesicles, insertion was monitored using a protease protection assay described previously (7, 18). The protected fragment (PF) diagnostic of proper insertion of the TA substrate is indicated.