Figure 2.

Molecular environment of arrested, laterally sorted precursor. (A–E) Mitochondria isolated from cells containing arrested cytb₂(1–147)DHFRHis (b₂147His) or no precursor (control) were incubated with the crosslinker DSG, solubilized with SDS and incubated with NiNTA-Agarose beads. Bound material was eluted with Laemmli buffer containing imidazole and analysed by SDS–PAGE and immunodecoration with antibodies to (A) Tim23, (B) Tim17, (C) Tim44, (D) Tim16 and (E) Tim14. Known crosslinking adducts are indicated. (F) ³⁵S-labelled cytb₂(1–147)DHFR was imported for 15 min into mitochondria in the presence of NADPH/DHF. Mitochondria were reisolated, resuspended in import buffer and incubated further. At the indicated time points, aliquots were removed, and either treated with proteinase K and directly analysed by SDS–PAGE and autoradiography (top panel) or first subjected to crosslinking with DSG followed by solubilization with SDS and IP with antibodies to Tim23, Tim44 or Tim14, as indicated, before analysis by SDS–PAGE and autoradiography (lower panels). b, bound fraction (100%); DSG, disuccinimidyl glutarate; i, intermediate form of imported protein; IP, immunoprecipitation; m, mature form of imported protein; mtHsp70, mitochondrial Hsp70; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); p, precursor form of imported protein; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; t, total (5%).