Fig. 4
From: The selectivity filter of the mitochondrial protein import machinery
Role of Tom70 glutamate residues 473, 542, and 577 in binding and import of AAC. a Location of glutamate residues E473, E542, and E577 in Tom70. The structural data were taken from Wu and Sha [53], PDB 2GW1. b Binding of [35S]-labeled AAC to Ni-NTA-bound wild type Tom70 (Tom70cd) or Tom70ΔGlu (E473Q/E542Q/E577Q), SD, n = 6. c Time course of binding of [35S]-labeled AAC to isolated yeast mitochondria containing either wild type Tom70 (Tom70WT) or Tom70ΔGlu, respectively. SD, n = 3. The relative amounts of mitochondrial Tom70 were assessed by immunoblotting (insert). d Import of AAC into isolated yeast mitochondria containing Tom70WT or Tom70ΔGlu, respectively. The mitochondria were incubated with [35S]-labeled AAC in presence of ATP at 25 °C and subsequently treated with proteinase K. The amounts of imported AAC in mitochondria containing Tom70WT within 10 min were set to 100%, SD, n = 3. e Mitochondrial import of F1β. The experiment followed the same procedure as in d. The amounts of imported F1β in mitochondria containing Tom70WT within 10 min were set to 100%, SD, n = 3. f Binding of AAC to mitochondria and subsequent import (binding and chase). Isolated mitochondria containing wild type Tom70 or Tom70ΔGlu were incubated with [35S]-labeled AAC in ATP-depleted reticulocyte lysate for 10 min at 25 °C, and reisolated, resuspended in buffer, and the suspensions were split into equal aliquots. The mitochondria were then again reisolated to determine the amounts of bound AAC (binding) or incubated in presence of 2 mM ATP and reisolated (chase), SD, n = 3