ChChd3 belongs to the subset of chch domain family proteins that are known to be primarily in the IMS of mitochondria and are characterized for their roles in protein import and as metallochaperones (22
). Although ChChd3 was identified in our laboratory a few years ago as a substrate for protein kinase A (15
), the function of this protein is so far completely unknown. In this study, we describe the characterization of ChChd3 as a novel protein essential for maintaining crista integrity and mitochondrial function. We provide evidence suggesting that ChChd3 is one of the key proteins that stabilizes protein complexes involved in maintaining crista architecture and protein import, the two primary processes that regulate mitochondrial structure and function.
Down-regulation of ChChd3 in HeLa cells resulted in fragmentation and clustering of the mitochondrial network (A
). Studies with the dominant negative mutant of Drp1, DRP1K38A
, which is resistant to fission, showed that the mitochondria in ChChd3 knockdown cells have impaired fusion activity. Additionally, ChChd3 interacts preferentially with the soluble IMS form of OPA1. Recent studies have suggested the essential role of OPA1 not only in mitochondrial fusion but also in remodeling of cristae (33
). Even though both the long and short isoforms have been shown to be essential in regulating mitochondrial fusion (42
) and crista morphology (10
), the individual role of these isoforms is not clearly understood. A recent immuno-EM study identified a differential distribution of the two yeast isoforms of OPA1 (Mgm1) across the IM (43
). Long Mgm1 was found strongly enriched in the crista membrane, and the short Mgm1 was preferentially localized on the OM/inner boundary membrane. Furthermore, by using various mutations in the GTPase domain, these authors showed that the functional GTPase domain is only required in short Mgm1 but not in long Mgm1 suggesting the idea of distinct function of each of these isoforms in mitochondria. It is likely that the fusion activity of OPA1 along with mitofusins on the OM is controlled by the s-OPA1 on the inner boundary membrane, whereas the crista morphology is regulated by long OPA1 on the crista membrane. Interaction of ChChd3 preferentially with soluble OPA1 and impaired fusion in ChChd3 knockdown cells indicate toward the potential involvement of ChChd3 in regulating the function of this isoform.
Depletion of ChChd3 resulted in major remodeling of the cristae and the CJ architecture ( and ). Previous studies have suggested that mitofilin and OPA1 are located at the CJs and involved in regulating crista morphology (12
). We demonstrate that ChChd3 interacts with mitofilin and OPA1, and loss of ChChd3 results in near loss of mitofilin and 50% reduction in OPA1 protein levels (C
). This suggests the possibility that all three proteins are in a complex at the CJs, where ChChd3 is a scaffolding protein that stabilizes the protein complexes and thus is involved in maintaining the integrity of CJs and in the formation/stabilization of cristae.
We also found that ChChd3 and mitofilin interact with Sam50 (), the component of the SAM complex on the OM, involved in β-barrel protein assembly. Very few proteins have so far been known to regulate both the protein import and organelle morphology (44
). The yeast protein, Mdm10, originally characterized for its role in mitochondrial dynamics and morphology has been shown to interact with SAM complex and is required for the β-barrel protein assembly (45
). To date, no mammalian proteins have been identified with dual functions in biogenesis of mitochondrial proteins and organelle morphology. Our results suggest the possible involvement of ChChd3 in protein import and/or assembly via SAM complex in addition to its role in regulating crista architecture.
The N-terminal POTRA domain of Sam50 is located in the IMS and has been shown to bind β-barrel precursor proteins in vitro
). In the IMS, ChChd3 may have a role in transporting the precursor proteins to the SAM complex. A similar function was suggested for IMS-translocase of inner membrane proteins (47
). However, so far there is no evidence showing the association of translocase of inner membrane proteins with the SAM complex.
As ChChd3 is localized to both the OM and IM fractions in the fractionated mitochondria and associates with both OM and IM proteins, we predict that ChChd3, together with mitofilin and Sam50, is located at contact sites where both the inner and outer membranes come together. These membrane contacts are known to be essential for various mitochondrial functions, including metabolite channeling, coordinated fusion, fission, protein transport, and also for maintaining structural integrity of outer and inner membranes (48
). Because CJs and contact sites are suggested to be two distinct structures on the IM and are not in preferential proximity to each other (49
), we propose that ChChd3 may be localized in discrete complexes at CJs and contact sites (I, model
). At the CJs, ChChd3 is likely to be associated with mitofilin and OPA1, and at the contact sites it is expected to be in a complex with mitofilin and Sam50. It is likely that ChChd3 is dynamically distributed between these two distinct complexes under different conditions in the mitochondria.
The distinct morphological changes observed due to the knockdown of ChChd3 are more severe than the knockdown phenotypes of mitofilin, OPA1, or MICS1, the mitochondrial proteins previously known to be involved in regulating crista morphology (11
). Loss of mitofilin resulted in an increase in the surface area of IM, with the cristae having degraded to the form of concentric sheets (12
). Down-regulation of OPA1 caused fragmentation of mitochondria and remodeling of cristae to reticular, curved, or ring-shaped structures (11
). The cristae in MICS1-depleted mitochondria were observed to be curved and ring-like invaginations of the IM (13
). In contrast, mitochondria in ChChd3 knockdown cells often did not contain any cristae. Those with cristae transitioned partly from lamellar to tubular cristae and overall showed a 50% reduction in the crista membrane surface area, indicating that either cristae were not formed properly or were disassembled more rapidly. Furthermore, loss of ChChd3 leads to a remarkable change in the CJ opening diameter without any significant changes in CJ number. This suggests that ChChd3 is not necessary for the formation of CJs; however, it is important for maintaining CJ architecture. On the contrary, loss of mitofilin was shown to affect the formation of CJs. Recent studies have established that CJ opening size depends on the OPA1 oligomers (10
), which are important to our finding that ChChd3 associates with OPA1 and are thus likely to have a role in OPA1 oligomerization and in maintaining CJ architecture. Consistent with the reduction in crista surface area, cells lacking ChChd3 also showed reduction in several IM protein levels. At this point, it is not clear if reduced crista content has triggered the reduction in IM proteins or the decreased crista content is the result of reduced levels of the proteins necessary for the proper formation and stabilization of the cristae.
The severe defect in mitochondrial morphology of ChChd3 knockdown cells is also reflected in strikingly lower rates of cellular respiration indicating a clear defect in mitochondrial function. Furthermore, the decrease in mitochondrial respiration is not compensated by an increase in glycolysis. It is likely that the deficiency of ChChd3 resulted in an overall reduced rate of substrate oxidation, energy production, and energy utilization that is consistent with the observed decreased rate of cell proliferation.
Our studies clearly suggest that ChChd3 is indispensable for maintaining crista structure and cellular metabolism. In a recent review, Perkins et al.
) compiled findings showing that the central nervous system has significantly greater crista to mitochondrial surface area compared with the peripheral nervous system. Accordingly, ChChd3 (referred to as FLJ20420), which we showed to be essential for crista integrity, is highly abundant at synaptic membranes and in the neurons of rat brain throughout the gray matter, spinal cord, and at the dorsal root ganglion (16
). Furthermore, recent studies have shown altered levels of ChChd3 in disease models of familial amyotrophic lateral sclerosis and ischemia (17
). Increasing lines of evidence have now indicated that the impairment of mitochondrial structure and function is one of the primary mechanisms behind motor neuron death in familial amyotrophic lateral sclerosis and cell death and injury in ischemia. Hence, it will be interesting to further understand whether ChChd3 plays a role in disease pathogenesis.
Our characterization of ChChd3 as a scaffolding protein for the SAM complex on the OM and mitofilin and OPA1 complex on the IM suggests that ChChd3 could be one of the key proteins that coordinate protein import and mitochondrial morphology, the two essential processes involved in proper functioning of mitochondria. Further studies are warranted to understand the dynamic localization of ChChd3 in these protein complexes and how ChChd3 regulates each of these protein complexes mechanistically. This will provide a better insight into the possible link between the essential processes that regulate organelle structure and function.