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. 2008 Mar 11;105(10):4022-7.
doi: 10.1073/pnas.0712209105. Epub 2008 Feb 22.

Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria

Christine Vande Velde  1 , Timothy M Miller, Neil R Cashman, Don W Cleveland
Affiliations

Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria

Christine Vande Velde et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in copper/zinc superoxide dismutase (SOD1) are causative for dominantly inherited amyotrophic lateral sclerosis (ALS). Despite high variability in biochemical properties among the disease-causing mutants, a proportion of both dismutase-active and -inactive mutants are stably bound to spinal cord mitochondria. This mitochondrial proportion floats with mitochondria rather than sedimenting to the much higher density of protein, thus eliminating coincidental cosedimentation of protein aggregates with mitochondria. Half of dismutase-active and approximately 90% of dismutase-inactive mutant SOD1 is bound to mitochondrial membranes in an alkali- and salt-resistant manner. Sensitivity to proteolysis and immunoprecipitation with an antibody specific for misfolded SOD1 demonstrate that in all mutant SOD1 models, misfolded SOD1 is deposited onto the cytoplasmic face of the outer mitochondrial membrane, increasing antigenic accessibility of the normally structured electrostatic loop. Misfolded mutant SOD1 binding is both restricted to spinal cord and selective for mitochondrial membranes, implicating exposure to mitochondria of a misfolded mutant SOD1 conformer mediated by a unique, tissue-selective composition of cytoplasmic chaperones, components unique to the cytoplasmic face of spinal mitochondria to which misfolded SOD1 binds, or misfolded SOD1 conformers unique to spinal cord that have a selective affinity for mitochondrial membranes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
A portion of SOD1 is localized to mitochondria enriched by buoyant density centrifugation. (A) Schematic outlining the enrichment of mitochondria according to buoyant density. (B) Demonstration of the selectivity of two SOD1 polyclonal antibodies used in this study. (C–F) SOD1 is enriched in floated mitochondria, compared with crude mitochondria and whole homogenates. SOD1 is enriched in spinal cord mitochondria and less so in cortical and liver mitochondria. VDAC, calnexin, and Hsp90 calnexin serve as markers for mitochondria, endoplasmic reticulum, and cytosol, respectively.
Fig. 2.
Fig. 2.
SOD1 associates with the cytoplasmic face of the outer mitochondrial membrane and correlates with disease. (A) Mitochondria enriched via floatation from spinal cord, cortex, and liver were treated with 100 μg/ml proteinase K (PK) in the presence or absence of 0.5% Triton X-100 (Tx-100). The majority of mitochondrially associated hSOD1 mutants with a partially misfolded conformation including hSOD1H46R (A), hSOD1G85R (B), and hSOD1G127X (D), are found deposited on the cytoplasmic face of the outer mitochondrial membrane in spinal cord as evidenced by sensitivity to proteolysis. In cortical mitochondria, the same mutants are completely insensitive to protease until addition of detergent, consistent with an intramitochondrial localization. Note that the absolute amounts of mutant SOD1 localized inside mitochondria is comparable in spinal cord and cortex. (C and D) The accumulation of mutant SOD1 protein on the surface correlates with disease because it is much less apparent in asymptomatic hSOD1G85R (C) and hSOD1G127X (D) animals. As expected, (A) hSOD1G93A and endogenous rat SOD1 (rSOD1) are not protease-sensitive. In all cases, the IMS protein Smac is only susceptible to proteolysis in the presence of detergent. Age is indicated in months. Data shown are representative of three independent experiments.
Fig. 3.
Fig. 3.
Mutant SOD1 is tightly bound to mitochondrial membranes in a salt-resistant manner. hSOD1H46R cannot be removed from isolated floated spinal cord mitochondria by extensive washing even with 1 M KCl. VDAC yields the expected pattern for an integral outer membrane protein. Data shown are representative of two experiments.
Fig. 4.
Fig. 4.
Mutant SOD1 is tightly bound to mitochondrial membranes in an alkali-resistant manner. (A) Schematic of alkali extraction. (B) Approximately one-third of hSOD1G93A, but almost no hSOD1WT protein, is tightly associated with spinal cord mitochondrial membranes in an alkali-resistant manner. In contrast, SOD1 is completely soluble in cortical mitochondria. (C) In contrast, hSOD1H46R, which adopts a partially unfolded conformation, is found in a higher proportion in spinal cord mitochondrial membranes compared with hSOD1G93A. (D and E) Time-dependent and disease-related integration of hSOD1G127X and hSOD1G85R into spinal cord mitochondrial membranes. VDAC and cytochrome c are used to identify behaviors of well defined mitochondrial components that are integral and peripheral membrane-associated, respectively. Data are representative of three experiments.
Fig. 5.
Fig. 5.
Mutant SOD1 associated with floated spinal cord mitochondria is misfolded. (A) The DSE2 epitope mapped onto hSOD1G37R crystal structure (Protein Data Bank ID code 1AZV), as generated by PyMol. (B) Schematic of immunoprecipitation experiments. (C) Floated isolated mitochondria from hSOD1H46R spinal cord and cortex were immunoprecipitated with two independent antibodies to the DSE2 region. Each uniquely identified a pool of misfolded SOD1 associated with spinal cord but not cortical mitochondria. (D and E) DSE2 recognizes a protease-sensitive pool of hSOD1G93A and hSOD1H46R but not hSOD1WT in solubilized (D) or intact floated spinal cord mitochondria (E), but not microsomal membranes.
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