High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease.

Purification of complex I variants from Y. lipolytica Complex

I was purified from Y. lipolytica strain GB30 carrying a deletion for the sulfur transferase (ST1) gene to enhance sample homogeneity ( 40 ). ST1 is a substoichiometric component of Y. lipolytica complex I, and the enzyme complex is fully functional in the absence of this polypeptide. Protein purification was carried out by His-tag affinity and size exclusion chromatography as described in ( 41 ), but on the Ni-NTA Sepharose column, the detergent was exchanged from 0.025% DDM to 0.025% LMNG. The complex I assembly intermediate accumulating in the Δ numm strain ( 34 ) (NUMM corresponds to NDUFS6; see table S2) was purified in the same way and polished by ion exchange chromatography on a MonoQ column. The complex I mutant from strain Δ nuym ( 12 ) (NUYM corresponds to NDUFS4; see table S2) was purified in the same way as the GB30 complex but without the detergent exchange. Activity measurements NADH:decylubiquinone (DBQ) oxidoreductase activity was measured in 20 mM Na + -Mops (pH 7.2), 50 mM NaCl, 2 mM KCN, and 0.025% LMNG using 150 μM DBQ and 100 μM NADH as substrates. The residual activity in the presence of 2 μM 2- n -decyl-quinazolin-4-yl-amine (DQA) was subtracted. For assignment of our preparation to the A or D form, we tested the sensitivity of complex I toward 5 mM MgCl 2 or 2 mM NEM. Activity measurements were carried out in 50 mM tris-HCl (pH 8.5), 80 mM KCl, 0.2 mM EDTA, 1.1 mM NaCN, and 0.025% LMNG with 150 μM DBQ and 100 μM NADH ( 9 ). Proton pumping assay with reconstituted complex I Asolectin (10 mg ml −1 ) from soybean was solubilized in 1.6% n -octyl glucopyranoside, 20 mM K + -Mops (pH 7.2), and 80 mM KCl. Purified complex I (200 μg) was reconstituted into proteoliposomes at a protein-to-lipid ratio of 1:50 (w/w) according to ( 42 ), but with 30-min intervals for the addition of BioBeads (Bio-Rad). Proteoliposomes were centrifuged at 100,000 g (1 hour, 4°C) and resuspended in 200 μl of 20 mM K + -Mops (pH 7.2) and 80 mM KCl. Proteoliposomes containing 5 μg of complex I were added to 2 ml of buffer [20 mM K + -Mops (pH 7.2), 50 mM KCl, and 0.5 μM valinomycin] in a stirred cuvette. Proton pumping was monitored by ACMA fluorescence quenching. The fluorophore was added to a final concentration of 0.5 μM. Measurements were performed in a Shimadzu RF-5001 fluorimeter at an excitation wavelength of 430 nm and an emission wavelength of 475 nm (bandpass, 5 nm each; integration time, 1 s) at 30°C. The reaction was started by the successive addition of 60 μM DBQ and 100 μM NADH. Last, 1 μM FCCP was added to collapse the pH gradient.

Mass spectrometry Purified complex

I from Y. lipolytica was denatured in 2 M guanidine hydrochloride. Proteins were digested with trypsin (sequencing grade, Promega) and analyzed by mass spectrometry using Thermo Scientific Q Exactive Plus equipped with an ultra-high performance liquid chromatography (UHPLC) unit (Thermo Scientific Dionex UltiMate 3000). Peptides and proteins (false discovery ratio,