*5.4. mitoCPR*

The mitochondrial compromised protein import response (mitoCPR) pathway was discovered in yeast and is activated when a mitochondrial protein is stalled in the Tom40 channel, inducing mitochondrial import stress and accumulation of proteins on the mitochondrial surface [376]. In yeast, the mitoCPR is activated and transcription factor Pdr3 induces the expression of *CIS1*. Cytosolic protein Cis1 binds to Tom70 and recruits the AAA+ ATPase Msp1, which removes stalled precursor proteins from mitochondrial channels and targets them for proteasomal degradation [376]. This allows mitochondria to maintain their functions under import stress conditions. This is interesting in the context of AD, especially given that APP, the precursor protein responsible for the production of toxic amyloid plaques in Alzheimer's brains, was shown to accumulate within TOM channels, driving mitochondrial dysfunction in AD [260]. This indicates that the mitoCPR pathway may be defective under these conditions, or may not be sufficient to rescue mitochondrial dysfunction associated with APP-TOM aggregation [377].

### *5.5. mitoTAD*

The mitochondrial protein translocation-associated degeneration (mitoTAD) pathway differs from those described already in that it is a quality control pathway that occurs constitutively under non-stress conditions [378]. In yeast, it is triggered by precursor proteins trapped in the Tom40 channel, sensed by Ubx2, which consistently interacts with the TOM complex under normal conditions, monitoring protein import through Tom40 [378]. If Ubx2 senses that a precursor protein is arrested within the TOM complex, a pool of Ubx2 binds to TOM and recruits the AAA+ ATPase Cdc48 for removal of arrested precursor proteins from the Tom40 channel [378]. The mitoTAD pathway was discovered in yeast, and interestingly, shows similarities to a quality control pathway in the ER, which involves Ubx2 exporting unfolded proteins from the ER [379,380]. No examples of the mitoTAD pathway have been described in models of neurodegeneration as of yet; however, as discussed above for the mitoCPR pathway, it is intriguing in the context of studies showing accumulation of proteins in the mitochondria during neurodegeneration, and further research into this link would be most interesting.

#### **6. Concluding Remarks**

Over recent years, remarkable progress has been made towards understanding the processes of mitochondrial protein import and respiratory complexes assembly. Recent advances in structural biology have begun to further elucidate the different structural properties of the mitochondrial translocases in high resolution, and this sheds further light on the various processes of mitochondrial protein import for specific protein classes. Whilst progress has been made, there remain areas of uncertainty regarding the organisation and dynamic action of the translocase complexes. Advances in import assay methods, such as the one recently developed [381], are also of paramount importance to dissect the mechanism of the import process and its kinetics. Hopefully, a revamped in cell assay will allow one to perform drug and phenotypic screenings, allowing for the easy identification of new players and modulators as well as small molecules that target this biological pathway.

Here, we highlight the body of evidence surrounding how closely interlinked mitochondrial protein translocation pathways are with the assembly of respiratory complexes and their function. Recent advances have begun clarifying exactly which translocation pathways are taken by nuclear-encoded respiratory complex proteins, though much more is yet to be done. Elucidating this link between import and respiratory function will be of vital importance, especially since in cases of mitochondrial disease, the importance of import pathways for respiratory complexes assembly and function is now clear. This suggests that targeting import pathways in cases of mitochondrial disease may become a credible therapeutic strategy.

Whilst mitochondrial dysfunction has long been recognised as a key factor in neurodegenerative diseases, mitochondrial protein import is now being implicated as a key factor in this dysfunction, across all levels of neurodegenerative disease models from the simple cell line setup right up to animal models and patient samples. Interestingly, the findings from these studies suggest that dysfunctional mitochondrial import is a driving force for the prominent mitochondrial irregularities observed in these diseases. This therefore represents an important target for further research to address the major outstanding questions. Namely, are the links between mitochondrial import defects and disease causative or consequential? What exact role might such defects play in disease progression?

This review has also outlined the various stress response pathways that have been shown to be activated in response to mitochondrial protein import defects. We highlight their importance in maintaining cell proteostasis and fine-tuning respiratory processes that rescue mitochondrial function. It is thought that these pathways are interlinked with one another; for example, the UPRam and UPRmt, the two most well characterised pathways to date, are thought to be activated simultaneously, despite having different triggers [364]. Interestingly, the UPRam and mPOS pathways both share a common trigger, that is, they become activated by accumulation of precursor proteins in the cytosol, yet thus far no evidence has shown their simultaneous activation. Interestingly, the mitoCPR and mitoTAD pathways also share the same trigger. It is thought that the mitoTAD pathway is active under non-stress conditions, whilst the mitoCPR pathway is activated only under stress conditions. Does this mean that mitoCPR is activated only when the mitoTAD pathway has failed? Since these stress response pathways are relatively new concepts, much more research is required. As more evidence emerges, enlightening the precise mechanisms of these pathways, they may generate therapeutically interesting targets for interventions against neurodegenerative diseases.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/life11050432/s1, Table S1: Supporting Table for Main Figure 3 on the Assembly of human Respiratory Complexes.

**Author Contributions:** Conceptualization, G.C.P. and H.I.N.; Writing, H.I.N., M.P. and G.C.P.; Figures, H.I.N., M.P. and G.C.P.; Revisions before submission, I.C. and J.P.; Revisions after submission, H.I.N., M.P., G.C.P. and J.M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** HN is supported by the Wellcome Trust Dynamic Molecular Cell Biology PhD programme (215317/Z/19/Z). MP is recipient of an MRC funded PhD scholarship. Research in JMH laboratory is supported the BBSRC (BB/R00787X/1), Wellcome Trust Investigator Award (220799/Z/20/Z), and Leverhulme Trust (RPG-2019-191). Research in the JP laboratory is supported by the Medical Research Council, UK (MC\_UU\_00015/7). Research in the IC laboratory is supported by the Wellcome Trust: Investigator Award (104632/Z/14/Z). GCP is supported by the Swiss National Science Foundation (Synergia project CRSII5\_180326). The APC was funded by University of Bristol Open Access via IC.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

∆ψ: mitochondrial transmembrane potential; α-syn: alpha-synuclein; Aβ: amyloid-beta peptide; AD: Alzheimer's Disease; AGK: acylglycerol kinase; AIF: apoptosis inducing factor; ALS: Amyotrophic lateral sclerosis; APP: amyloid precursor protein; CCCP: carbonyl cyanide-*m*-lorophenylhydrazone; CHCHD: coiled-coil-helix-coiled-coil-helix domain; CI-V: mitochondrial respiratory complexes one to five; cryo-EM: cryogenic electron microscopy; DAMPs: damage-associated molecular patterns; Drp1: mitochondrial fission GTPase dynamin-related protein 1; eIF2α: eukaryotic translation initiation factor 2 alpha; ER: endoplasmic reticulum; ETC: electron transport chain; Fe/S: iron/sulphur; HD: Huntington's disease; HSR: heat shock response; Hsp: heat-shock protein; HTT: Huntingtin; IBM: inner boundary membrane; IMM: inner mitochondrial membrane; IMS: intermembrane space; iMTS: internal mitochondrial targeting sequence like signal sequence; ISR: integrated stress response; LHON: Leber hereditary optic neuropathy; MCIA: mitochondrial complex I intermediate assembly; MCSR: mitochondrial to cytosolic stress response; MDVs: mitochondrial-derived vesicles; MIA: mitochondrial IMS assembly; MICOS: mitochondrial contact site and cristae organising system; MIM: insertase of the outer mitochondrial membrane; MIP: mitochondrial intermediate peptidase; mitoCPR: mitochondrial compromised protein import response; mitoTAD: mitochondrial protein translocation-associated degeneration; MITRAC: Mitochondrial Translation Regulation Assembly intermediate of Cytochrome *c* oxidase; mPOS: mitochondrial precursor over-accumulation stress; MPP: mitochondrial processing peptidase; mtDNA: mitochondrial DNA; MTS: mitochondrial targeting sequence; NFTs: neurofibrillary tangles; NLS: nuclear localisation sequence; OMM: outer mitochondrial membrane; OXA1: mitochondrial oxidase assembly protein 1; PAM: presequence translocase-associated motor; PD: Parkinson's disease; ROS: reactive oxygen species; SAM: sorting and assembly machinery; SC: supercomplexes; *SN*: *Substantia Nigra*; TIM22: translocase of the inner membrane 23; TIM23: translocase of the inner membrane 23; TOM: translocase of the outer membrane; TOM-CC: translocase of the outer membrane core complex; UQCC1 and UQCC2: ubiquinol-cytochrome *c* reductase complex assembly factors 1 and 2; UPRam: UPR activated by the mistargeting of proteins; UPRmt: mitochondrial unfolded protein response; UTR: untranslated region.

#### **References**

