Fig 3. The Sec61p N-terminal helix is important for post-translational protein import into the ER in vivo.

A: Co-translational ER import of newly synthesized DPAPB in sec61S2Y (left panel), sec61ΔH1, and sec61ΔN21 (right panel). Cells were grown at 30°C to early exponential phase, labeled with ³⁵S-Met/Cys for 5 min and lysed. Cytosolic precursor (pDPAPB) and ER-membrane integrated DPAPB were immunoprecipitated, resolved by SDS-PAGE, and detected by autoradiography. B: Post-translational ER import of ppαF in sec61S2Y (left panel), sec61ΔH1, and sec61ΔN21 (right panel). Cells were grown at 30°C to an OD600 = 1 and shifted to the indicated temperatures for 3h, extracts were prepared by bead-beating, and samples resolved by SDS-PAGE. Proteins were transferred to nitrocellulose and the accumulation of cytosolic ppαF was analyzed by immunoblotting. C: Post-translational ER import of newly synthesized pCPY* in sec61S2Y (left panel), sec61ΔH1, and sec61ΔN21 strains (right panel). Cells were grown at 30°C to early log phase, labeled with ³⁵S-Met/Cys for 5 min and lysed; cytosolic pCPY* and ER-lumenal CPY* were immunoprecipitated, resolved by SDS-PAGE and detected by autoradiography. Duplicate samples were taken for experiments shown in panels A, B (right), and C. Experiments were performed three times.