Respiratory supercomplexes – assembly and stabilizing factors

Although the role and function of the respiratory supercomplexes remains ill-defined, all theories have in common that they indicate an essential role in proper mitochondrial function.

Given the high consistency in particles obtained by cryo-EM in different laboratories (Althoff et al., 2011; Dudkina et al., 2011; Mileykovskaya et al., 2012; Hartley et al., 2019, 2020; Rathore et al., 2019) and therefore largely uniform population of supercomplexes, it seems obvious that the coordination underlies a specific mechanism. Yet, not completely resolved, several proteins/factors were found to be involved in supercomplex assembly additionally considering that the organization might be different from yeast to mammals.

Cardiolipin. The inner mitochondrial membrane is mainly composed of the lipids phosphatidylcholine, phosphatidylethanolamine and cardiolipin. The latter is found exclusively in mitochondria and was proven to be a key player in supercomplex stabilization (Zinser et al., 1991; Schenkel and Bakovic, 2014; Rappocciolo and Stiban, 2019).

However, it is discussed if cardiolipin is only responsible for supercomplex stabilization or if it takes part in the formation itself (Pfeiffer et al., 2003; Bazán et al., 2013). Several cryo-EM structures demonstrated that cardiolipin is integrated into yeast supercomplex III2IV(1-2) and recently it was even resolved at the interaction site between complex IV subunit Cox5 and complex III subunits Rip1 and Qcr8 (Mileykovskaya et al., 2012; Hartley et al., 2019, 2020;

Rathore et al., 2019). Furthermore, the right balance between cardiolipin and phosphatidylethanolamine with the inner mitochondrial membrane appears to be important since they showed opposing features. Absence of phosphatidylethanolamine leads to a more stable supercomplex in yeast but causes similar defects in respiration and membrane potential (Böttinger et al., 2012). Defects in human cardiolipin synthesis cause heavily compromised mitochondrial structures and manifests in a disease called Barth syndrome. Patients with Barth syndrome suffer from multi-system disorder, first characterized as cardiac disease, emphasizing once more the physiological importance of supercomplex stability (reviewed in Clarke et al., 2013).

SCAF1 (COX7A2L). SCAF1 is expressed in higher eukaryotes and has no yeast homolog. It was first identified due to its high sequence similarity to COX7 isoforms, therefore the name COX7A2L (Lapuente-Brun et al., 2013). Further investigations characterized it as a possible assembly factor of supercomplexes containing complex III and complex IV, since it was only present in those but not the single complexes (reviewed in Lobo-Jarne and Ugalde, 2018).

Although initially reported as stabilizing complex IV at the site of complex I (Ikeda et al., 2013;

Lapuente-Brun et al., 2013), other studies argued that it is not essential for respirasome (I-III2-IVn) assembly (Pérez-Pérez et al., 2016). Only the association of complex III and complex IV appears to be affected. Lobo-Jarne et al. (2018) suggested that SCAF1 is involved in a check-point step of complex III assembly and furthermore demonstrated that knock-out cells did not suffer from dysfunctional respirasomes but delayed assembly. This lines up with various observations with mouse models expressing the putative non-functional shorter isoform (Mourier et al., 2014; Davoudi et al., 2016). Although this questions the physiological relevance for complex III2IV(1-2), it indicates that complex I is able to serve as a scaffold in the respirasome

but that the association between complex III and complex IV is favorable in terms of efficient supercomplex assembly and metabolic fitness (García-Poyatos et al., 2020).

Coi1. Coi1 is conserved among fungi and was identified by Singhal et al. (2017) as a transient interactor of complex IV. Mutant strains display defective supercomplex assembly while heme insertion into Cox1 appears to be affected in parallel. Coi1 does not directly bind to heme or Cox1, yet, it seems to facilitate heme incorporation into complex IV (Singhal et al., 2017).

Consequently, it is elusive if supercomplex assembly is directly affected. However, several complex III and complex IV subunits and the Rcf-proteins (see below) were found in its interaction spectrum indicating an involvement in supercomplex assembly and/or stabilization (Singhal et al., 2017).

Rcf-proteins. Rcf1, Rcf2 and Rcf3 share homologous sequences among each other and were characterized as possible assembly factors of respiratory supercomplexes. All three proteins are independently interacting with complex III and complex IV, while predominantly associating via the supercomplex III2IV(1-2) in a substoichiometric manner (Chen et al., 2012; Strogolova et al., 2012; Vukotic et al., 2012; Römpler et al., 2016). Rcf1 appears to be involved in Cox3 modular assembly, however, it remains associated at the supercomplex and facilitates Cox13 and Rcf2 association (Strogolova et al., 2012; Vukotic et al., 2012; Garlich et al., 2017). While supercomplex formation is not completely abolished in rcf1∆ but strongly affected, the idea of a true assembly factor was questioned (Chen et al., 2012; Vukotic et al., 2012; Strogolova et al., 2019; Dawitz et al., 2020). Rcf2 and Rcf3 in turn, were demonstrated to have overlapping roles in respect of complex IV regulation (Römpler et al., 2016). It was intriguing when Rcf2 was resolved in a recent cryo-EM structure of allegedly fully assembled hypoxic supercomplex III2IV arguing for a stoichiometric interaction under these conditions (Hartley et al., 2020).

Rcf2 and Rcf3 are conserved among fungi, whereas Rcf1 possesses two mammalian homologs the hypoxia inducible HIGD1A and the constitutively expressed HIGD2A (Timón-Gómez et al., 2020b). HIGD2A was first reported as the functional homolog of Rcf1 and appears to be involved in COX3 module maturation and its assembly into complex IV (Chen et al., 2012;

Vukotic et al., 2012; Hock et al., 2020). Nevertheless, Timón-Gómez et al. (2020a) recently found HIGD2A to be involved in supercomplex assembly, while it displays overlapping functions with HIGD1A in complex IV biogenesis. HIGD1A in turn, was demonstrated to play an additional role in complex III2 biogenesis (Timón-Gómez et al., 2020a).