Fig 2: Proteinase K protection assay for wild-type and mutant EndoG and SOD1.a, b, Wild type and mutant forms of EndoG (a) and SOD1 (b) were transfected in HeLa cells and analyzed for their mitochondrial import. Isolated mitochondria were resuspended in a hypotonic (osmotic swelling) buffer to generate mitoplasts. Proteinase K (10 µg/ml) was added to digest accessible proteins. Samples were analyzed by SDS-PAGE followed by immunoblotting using anti-GFP (EndoG or SOD1), anti-TOM20 (OM), anti-AIFM1 (IMS), anti-HCCS (IM), and anti-HSP60 (matrix) antibodies. Triton-X100 was added to solubilize matrix proteins and facilitate their proteinase K digestion, which revealed that mutant SOD1-G93A is partially protease-resistant as it is poorly digested even upon solubilization of all the different mitochondrial compartments. Results are representative of two replicates (a,b). Unprocessed blots are available in source data. Source data

Fig 3: Overexpression of untagged small heat shock proteins demonstrates that a fraction of most family members reside in the mitochondrial intermembrane space.Proteinase K protection assay on mitochondria isolated from non-heat shocked HeLa cells that were transiently transfected with untagged small heat shock proteins. This allowed us to assess the mitochondrial localization of tissue-specific sHSPs like HSPB4 (expressed in the eye lens) and HSPB9 (expressed in the testis). Isolated mitochondria were resuspended in a hypotonic (osmotic swelling) buffer to generate mitoplasts. Proteinase K (10 µg/ml) was added to digest accessible proteins. Samples were analyzed by SDS-PAGE followed by immunoblotting using anti-HSPB antibodies and anti-TOM20 (OM), anti-AIFM1 (IMS), anti-HCCS (IM), and anti-SDHA (matrix) antibodies. Results are representative of two replicates. Unprocessed blots are available in source data. Source data

Fig 4: Small heat shock proteins detach from mitochondrial membranes around pH 11.5 and have no effect on mitochondrial membrane potential.a, Membrane association of small heat shock proteins was verified with a sodium carbonate assay. This assay allows the separation of integral membrane proteins (retained in the pellet) from peripheral membrane proteins and soluble proteins (extracted into the supernatant). Mitochondria isolated from HeLa cells were resuspended in Na2CO3 set to the indicated pH. Samples were analyzed by SDS-PAGE followed by immunoblotting using anti-HSPB1, anti-HSPB5, anti-HSPB8, anti-CytC (IMS), anti-mtHSP60 (matrix), anti-AIFM1 (IMS), anti-TOM20 (OM), anti-HCCS (IM), and anti-TIM23 (IM) antibodies. T = total; P = pellet; S = supernatant. Unprocessed blots are available in source data. b, Flow cytometry gating strategy and TMRE membrane potential in control or sHSP-depleted HeLa cells. As a negative control, the membrane potential was dissipated by CCCP treatment. Related to Fig. 3d: ungated, non-debris, singlets, TMRE-intensity. FSC: forward scatter, SSC: side scatter, A: Area, H: height, W: width. Results are representative of one (b), in which more than 130,000 cells were counted per condition, or three (a) replicates. Unprocessed blots are available in source data. Source data

Fig 5: BAG3 is recruited to mitochondria upon heat shock but is not imported into mitochondria.a, HeLa cells were subjected to heat shock (42 °C) for 1 h. Cells were collected afterward and the mitochondrial fraction was separated from the cytosolic fraction. Using immunoblotting we verified the abundance of BAG3 and sHSPs in each fraction. Tubulin was used as a marker for the cytosol and VDAC1 was used as a marker for mitochondria. The NP-40-soluble whole cell lysate (WCL) was used as a reference. b, Proteinase K protection assay on mitochondria isolated from HeLa cells. Isolated mitochondria were resuspended in a hypotonic (osmotic swelling) buffer to generate mitoplasts. Proteinase K (10 µg/ml) was added to digest accessible proteins. Samples were analyzed by SDS-PAGE followed by immunoblotting using anti-BAG3, anti-TOM20 (OM), anti-AIFM1 (IMS), anti-HCCS (IM), and anti-SDHA (matrix) antibodies. c, BAG3 knockout HeLa cells were rescued with wild type or delta-IPV BAG3-GFP (deficient in binding to HSPB8) and subjected to proteinase K protection assay as in panel B. Samples were analyzed by SDS-PAGE followed by immunoblotting using anti-BAG3, anti-TOM20 (OM), anti-AIFM1 (IMS), anti-HCCS (IM), and anti-HSP60 (matrix) antibodies. Results are representative of two (a,b) or three (c) replicates. Unprocessed blots are available in source data. Source data