Mitochondrial accumulation of amyloid β (Aβ) peptides requires TOMM22 as a main Aβ receptor in yeast

Abstract

Mitochondrial accumulation of intracellular β-amyloid (Aβ) peptides is present in the brains of individuals with Alzheimer's disease (AD) as well as in related mouse models of AD. This accumulation is extremely toxic because Aβ disrupts the normal functions of many mitochondrial proteins, resulting in significant mitochondrial dysfunction. Therefore, understanding the mitochondrial accumulation of Aβ is useful for future pharmaceutical design of drugs to address mitochondrial dysfunction in AD. However, the detailed molecular mechanism of this accumulation process remains elusive. Here, using yeast mitochondria, we present direct experimental evidence suggesting that Aβ is specifically recognized by translocase of outer mitochondrial membrane subunit 22 (Tom22 in yeast; TOMM22 in human), a noncanonical receptor within the mitochondrial protein import machinery, and that this recognition is critical for Aβ accumulation in mitochondria. Furthermore, we found that residues 25-42 in the Aβ peptide mediate the specific interaction with TOMM22. On the basis of our findings, we propose that cytosolic Aβ is recognized by TOMM22; transferred to another translocase subunit, TOMM40; and transported through the TOMM channel into the mitochondria. Our results not only confirm that yeast mitochondria can be used as a model to study mitochondrial dysfunction caused by Aβ peptides in AD but also pave the way for future studies of the molecular mechanism of mitochondrial Aβ accumulation.

Keywords: Ni-NTA conjugated nanogold; Tom22; amyloid-β (Aβ); chromatography; electron microscopy (EM); mitochondria; protein import; protein-protein interaction; yeast.

Figure 1.

Yeast mitochondria specifically recognize and accumulate human Aβ peptides. A, illustration of the import and stop experiment. The experiment was started by adding the recombinant protein His-GST-Aβ or His-GST into the isolated mitochondrial solution and stopped in 20 min by centrifugation. The treated mitochondria were then washed thoroughly before Western blot analysis and nanogold labeling. B, Western blot analysis of the treated mitochondria from A probed by the anti-GST antibody. The left panel shows the total input proteins His-GST-Aβ and His-GST (as a control) before the wash, and the right panel shows significant accumulation of His-GST-Aβ but not His-GST after the wash. Yeast Sce1 is the loading control for all Western blot analysis in this work. C, quantification of each band in B. Data of three independent experiments are shown. *, p < 0.01 (two-tailed Student's t test, compared with His-GST after wash). D and E, representative EM images of the nanogold-labeled mitochondria treated with His-GST (D) and His-GST-Aβ (E). The arrow indicates labeled nanogold. The scale bar in both micrographs is 200 nm.

Figure 2.

Mitochondrial Aβ accumulation is TOM-dependent. A, illustration of the experiment. Yeast mitochondria were preloaded with preADHIII substrates and then incubated with His-GST-Aβ for the import and stop assay. B, Western blot analysis of the treated mitochondria. The mitochondria were pretreated with or without preADHIII-GST and then treated with or without His-GST-Aβ. The left two lanes used anti-GST probe, and the right two lanes used anti-His probe. C and D, representative EM micrographs of the nanogold-labeled mitochondria that were either pretreated with preADHIII-GST-His (C) or treated with both preADHIII-GST and His-GST-Aβ. The scale bar is 200 nm.

Figure 3.

Neither Tom20 nor Tom70 binds to Aβ peptides in vitro. A, cytoTom20, cytoTom70, His-GST-Aβ, and preADHIII-GST were purified by gel filtration on a Superdex 200 column, respectively, in buffer containing 20 mm HEPES, pH 7, 150 mm NaCl. The peaks, from left to right, are cytoTom70 (∼13.7 ml), preADHIII-GST (tall, ∼15 ml), His-GST-Aβ (short, ∼15 ml), and cytoTom20 (∼16.2 ml). B, C, and D, mixtures of preADHIII-GST–cytoTom20 (1:3 molar ratio), His-GST-Aβ–cytoTom20 (1:3), and His-GST-Aβ–cytoTom70 (2:1) were examined by analytical gel filtration, respectively. Abs, absorbance; mAU, milli-absorbance units.

Figure 4.

Tom22 is responsible for the Aβ peptides binding. A, gel filtration chromatography of the purified cytoTom22. B, the mixture of His-GST-Aβ–cytoTom22 (1:3) was examined by analytical gel filtration. C, Western blot analysis of the substrate accumulation by isolated mitochondria from 22T and 20T strains with and without TEV protease digestion. D, Western blot analysis of the mitochondrial Aβ accumulation using antibody-shielded mitochondria. E, mitochondrial Aβ accumulation under competitive experiments with purified cytosolic domains of Tom20, Tom70, or Tom22. F, mitochondrial accumulation of various GST-tagged Aβ fragments tested in the import and stop experiments. Abs, absorbance; mAU, milli-absorbance units.