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. 2016 Nov 1;27(21):3257–3272. doi: 10.1091/mbc.E16-05-0313

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© 2016 Cenini et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — Mitochondrial import steps affected by Aβ peptides. (A) Binding of the precursor protein to the OMM import machinery receptors. After removing the Δψ_mt, mitochondria were incubated for short times (range of seconds) with Aβ peptides (3.5 μM) and precursor protein [³⁵S]Su9(70)DHFR. Half of the samples were incubated with PK (50 μg/ml) to digest nonimported precursor protein. (B) Separation of preprotein binding (Binding) to OMM from inner membrane translocation and processing steps (Chase). For precursor binding and insertion into the OMM, Δψ_mt was dissipated by CCCP (1 μM) during incubation with [³⁵S]Su9(70)DHFR in the presence (lanes 11 and 12) and absence (lanes 10 and 13–18) of Aβ peptides. To assay inner membrane translocation and processing (Chase), Δψ_mt was restored by addition of BSA (2 mg/ml; lanes 10–12 and 16–18) in the presence (lanes 17 and 18) and absence of Aβ peptides. For comparison, a complete one-step import reaction was performed (lanes 1–9). (C, D) Effect of Aβ peptides on cotranslational and posttranslational import. (C) As shown in the scheme of the experimental setup, cotranslational import was performed by incubating rabbit reticulocyte lysate, Su9(70)DHFR mRNA, [³⁵S]methionine, and isolated energized mitochondria in the presence or absence of Aβ peptides (3.5 μM) as indicated at 30°C for 60 min. (D) In posttranslational import, first the translation of [³⁵S]Su9(70)DHFR was performed using rabbit reticulocyte lysate, Su9(70)DHFR mRNA, and [³⁵S]methionine for 30 min, and then isolated mitochondria were added in the presence of Aβ peptides (3.5 μM) for an additional 30 min to perform the mitochondrial import reaction. The translation was stopped by adding 8 mM cold methionine. All samples were analyzed by tricine-SDS–PAGE, followed by Western blot, digital autoradiography, and immunodecoration against Aβ peptides and Tim23. L, total amount of Aβ peptides added; m, mitochondrial mature form; Mock, control experiment in the absence of mitochondria; p, mitochondrial precursor protein; WB, Western blot.