Figure 7.

The NH₂-terminal domain of Tom40 is essential for correct assembly. (A) The Tom40ΔN and the Tom40ΔC precursors were incubated with isolated mitochondria from normal culture or from a culture to which CHX (150 μg/ml) was added 90 min before isolation of mitochondria (+CHX). After incubation for 20 min at 25°C the mitochondria were reisolated and one sixth of the material was loaded on the gel (Total). The rest was solubilized with 0.5% digitonin, halved, and subjected to immunoprecipitation with the indicated antibodies or with antibodies from preimmune serum (PIS). (B) Tom40ΔN cannot be fully assembled into the Tom complex. Radiolabeled precursors were incubated with intact mitochondria for 20 min at 25°C. Further treatment and analysis by BNGE was as described in Fig. 5 C. Fully assembled material and the high molecular intermediate are indicated by A and I, respectively. (C) Tom40ΔN is not stably associated with Tom22 and Tom6. Tom40 and Tom40ΔN precursors were incubated with mitochondria for 20 min at 25°C and mitochondria were reisolated. One aliquot was directly analyzed by SDS-PAGE, the rest was solubilized with 0.5% digitonin, split into three aliquots which were subjected to immunoprecipitation with antibodies against Tom22, or Tom6, or with antibodies from preimmune serum (PIS). PIS could not precipitate the precursors (data not shown).