A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

Abstract

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

Figure 1. Exposure of native complex I to LDAO generates subcomplex Iδ

(A) BN-PAGE of solubilized mitochondrial membranes from Y. lipolytica (left lane) and subcomplex Iδ (right lane). The migration behavior of subcomplex Iδ (Iδ) indicated a lower mass compared with native complex I (CI). Note that complex I subunit ST1 is detached under BN-PAGE conditions resulting in a total mass of ~930 kDa for the control; complex V monomer (CV, ~580 kDa), complex III dimer (CIII₂, ~420 kDa). The bands in the high molecular weight range in the right lane were identified as oligomers of subcomplex Iδ (n Iδ). (B) Subcomplex Iδ (light grey) eluted as a symmetrical peak with slightly increased retention volume compared with native complex I (black) in size exclusion chromatography. (C) Non-destructive LILBID-MS analysis of subcomplex Iδ at low laser intensity. A prominent peak series indicated m/z values (Iδ + ND2, blue) of ~ 850 kDa including detergent and lipids. As for native complex I the protein mass was found to be 40–50 kDa offset from the peak maximum and best approximated by evaluating m/z values of the left peak edges the mass of subcomplex Iδ + ND2 was determined to be ~808 kDa (blue ticks). A second peak series corresponding to ~755 kDa was assigned to subcomplex Iδ without ND2 (Iδ, red ticks). A third peak series of ~413 kDa can be assigned to the peripheral arm (Q/N-module, green ticks) that is generated by disintegration of laser irradiation sensitive subcomplex Iδ (red) (see text for details).

Figure 2. EPR spectra of complex I and subcomplex Iδ

Spectra were recorded using the following parameters: microwave frequency 9.47 GHz, modulation amplitude 0.64 mT, modulation frequency 100 kHz. Complex I or subcomplex Iδ (15 mg/ml) were reduced with NADH (2 mM) and the spectra were recorded at 12 K. *Organic radical signal, probably representing an impurity.

Figure 3. dSDS-PAGE of purified complex I (A) and subcomplex Iδ (B)

Subunits missing in subcomplex Iδ are highlighted by a circle. In the subcomplex an extra spot was visible near the 49-kDa subunit (49-kDaΔN). It was identified by ESI-MS/MS as a degradation product of the 49-kDa subunit lacking a 3 kDa fragment at the N-terminus. Inserts 1 and 2 show the section of a dSDS-gel around subunit NUJM of complex I or subcomplex Iδ prepared from a strain carrying a strep-tagged version of this subunit. Insert 3 shows a section of a Coomassie-stained BN-dSDS-PAGE gel of complex I (left) and the corresponding Western blot (right). Subunit NUPM was recognized by monoclonal antibody (mab) 31A8 (anti-NUPM). Note that the assignment for some of the accessory subunits was updated and is therefore different from [22].

Figure 4. LILBID mass fingerprint spectra of complex I and subcomplex Iδ

The peaks are numbered according to Table I. Insert, detail from mass spectra of a complex I variant carrying a strep-tag on subunit NUJM (grey, dashed line) and the corresponding subcomplex Iδ (black, dashed line). The mass shift permitted to identify subunits NUJM (18) and ND6 (13) in the subcomplex (asterisks). Taking into account new evidence on C-terminal peptides and prediction of targeting sequences (Table I) we reassigned the peaks for subunits NUMM and NI9M (compare [8]). The peak at 7.7 kDa previously assigned to NI9M remains unassigned. To account for these changes and the newly discovered subunits NUUM and NEBM, the assignment of peaks 27 to 29 and peaks 37 to 42 was updated to match decreasing molecular masses of the corresponding subunits. The spectrum of subcomplex Iδ exhibited a few additional peaks that correspond either to higher charged species of the 75-kDa (1), 51-kDa (2) and 49-kDa (3) or to the truncated version of the 49-kDa subunit (3ΔN, see Figure 3).

Figure 5. Subunit arrangement in complex I (A) and subcomplex Iδ (B) from Y. lipolytica

Accessory subunits form an extended scaffold and connect the peripheral arm (Q/N-module, white) and the membrane arm (P_P-module, light grey; P_D-module, dark grey). See text for details. The long helical transmission element [11] implicated in conformational energy transfer is shown in light grey. In the subcomplex subunit ND2 is shown transparent, because it was present in substoichiometric amounts. Subunit ST1 is not shown. The positions of subunits NB4M, NIDM and NI2M have not been confirmed experimentally.

Figure 6. Localization of the NUPM subunit on the intermembrane space side of the membrane arm

Two-dimensional average of single particle images of complex I decorated with monoclonal antibody 31A8 against the NUPM subunit. Alignment and classification of 749 images yielded six major classes. Class 3, shown here and containing 16% (118) of the particles, most clearly shows the bound antibody. The resolution of the average is 30 Å as measured by the Fourier Ring Correlation with a cutoff criterion of 0.3[53]. Scale bar, 100 Å.

Figure 7. Membrane arm of mitochondrial complex I

Section from electron density map (blue mesh) of Y. lipolytica complex I and structural model for transmembrane segments (yellow) [11]. The contour line of a complex I monomer is tentatively indicated by a dashed blue line; the red line separates P_P and P_D-module; DMP, distal membrane arm protrusion. A prominent continuous layer of extrinsic protein mass is attached to the intermembrane space side (IMS) of the membrane arm (compare Fig. 5).