The Assembly Pathway of Mitochondrial Respiratory Chain Complex I

Abstract

Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex.

Functional asymmetry and electron flow in the bovine respirasome

“…These observations are in agreement with our 8.1 Å global average (Figure 4C,D), in which the density for the 51 kDa subunit is weak and could explain why the NADH:dehydrogenase module is absent in several of the 3D class averages (Figure 1—figure supplement 2). However, other studies suggest that supercomplexes form by association of fully assembled component complexes (Guerrero-Castillo et al, 2016), which would thus not account for the intermediate structures we observe.…”

“…In line with this, a model for the generation of supercomplexes was proposed, where the assembly of catalytic subunits of the complex I NADH:dehydrogenase module occurs at a late stage to activate the supercomplexes (Moreno-Lastres et al, 2012). However, recent complexome profiling studies failed to detect supercomplexes containing immature complex I, suggesting that the respirasome forms by association of fully assembled component complexes (Guerrero-Castillo et al, 2016). …”

Kinetics and Mechanism of Mammalian Mitochondrial Ribosome Assembly

“…Coordinated assembly appears to be necessary to recruit eight proteins that bind at early stages in mitoribosome assembly but lack substantial contact with rRNAs, including mS22, mS31, mS35, mS39, bS1m, mL39, mL50, and mL45. Mitoribosome assembly would be facilitated by association of MRPs in pre-assembled sub-complexes, as has been documented for assembly of mammalian respiratory complex I (Guerrero-Castillo et al, 2017). While this is not absolutely required for coordinated assembly, the model generated by our SILAC labeling approach provides a guide for future experiments to search for such intermediates and to dissect the assembly sequence in greater detail.…”

“…Coordinating assembly of a mitoribosome is clearly a daunting task, since it requires import of as many structural proteins as the entire respiratory chain. We know that the assembly of a simpler structure, NADH dehydrogenase, requires nearly 24 hr for the 44 component proteins to join the complex (Guerrero-Castillo et al, 2017; Ugalde et al, 2004). The kinetics of mitoribo-some assembly may be limited by the time required to import the 82 individual MRPs into mitochondria.…”

Regulation of Mitochondrial Complex I Biogenesis in Drosophila Flight Muscles

“…A current hypothesis is that the accessory subunits may regulate ROS formation, complex assembly or stability, and cellular homeostasis in vivo . Of note, disease-causing mutations in several accessory subunits have been identified (Berger et al, 2008; Budde et al, 2000; Hoefs et al, 2008; Hoefs et al, 2011; Kirby et al, 2004; Ostergaard et al, 2011; Scacco et al, 2003); and genetic disruption of some accessory subunits in cell lines impair CI assembly (Guerrero-Castillo et al, 2017; Stroud et al, 2016). However, a definitive role for many of the accessory subunits in vivo remains to be established.…”