Skd3 (human CLPB) is a potent mitochondrial protein disaggregase that is inactivated by 3-methylglutaconic aciduria-linked mutations

Ryan R. Cupo, James Shorter

Preprint posted on 18 January 2020

Article now published in eLife at

Long-lost mitochondrial disaggregase has been found

Selected by Tessa Sinnige

Categories: biochemistry


Protein aggregation is a challenge for any living cell, resulting in the loss of protein function and the generation of potentially toxic misfolded protein species. Non-metazoan eukaryotes such as yeast employ specialized molecular chaperones, Hsp104 in the cytosol and nucleus and Hsp78 in mitochondria, that are capable of untangling protein aggregates, allowing the native protein state to be restored. Curiously, these disaggregases seem to have been lost during evolution and are absent in metazoa such as ourselves. Recently, human chaperone complexes capable of disaggregation in the cytosol were identified (1, 2), but a mitochondrial disaggregase so far remained elusive. In this preprint, the authors investigate whether Skd3, an AAA+ ATPase that has some resemblance to Hsp78 and Hsp104, is in fact the long-sought mitochondrial disaggregase in metazoa.


The authors find that Skd3…

  • … has ATPase and disaggregase activity. Using in vitro assays, the authors show that purified Skd3 causes aggregated luciferase to partially refold, and that this function is dependent on its ATPase activity. Mutation of a residue in the conserved pore loop that is thought to be critical for substrate binding abolishes the disaggregase activity of Skd3.


  • … is regulated by an auto-inhibitory peptide. Skd3 contains a hydrophobic N-terminal stretch that is cleaved by the inner membrane protease PARL. When the authors express Skd3 without this region, mimicking PARL cleavage, they find that the disaggregase activity of Skd3 is boosted by over 10-fold compared to the full-length protein.


  • … is capable of disaggregating α-synuclein fibrils. The authors note that the requirements for disaggregating amorphous aggregates versus highly structured amyloid fibrils may differ. However, using a sedimentation assay combined with a dot blot they observe that Skd3 can disaggregate α-synuclein fibrils in the presence of ATP.


  • … disease mutations impair the function of the protein. Mutations in Skd3 are associated with the rare mitochondrial disorder 3-methylglutaconic aciduria, type VII (MGCA7). When testing four Skd3 mutants in their in vitro assay, the authors find that the ATPase activities vary but the disaggregase activity correlates with disease severity. Altogether the authors conclude that the function of Skd3 as a mitochondrial disaggregase is crucial for human health.


Figure 9 from the preprint showing the mitochondrial disaggregase Skd3 in action. Reproduced with permission from the authors.


Why I chose this preprint

As the authors nicely outline in their introduction, it has so far remained a mystery how metazoa can do without the disaggregase proteins Hsp104 and Hsp78 that are present in yeast, but were lost during evolution. Machineries capable of disaggregating amorphous and fibrillar aggregates in the metazoan cytosol have been identified in recent years (1, 2) (see also my recent preLight on this topic), but a mitochondrial disaggregase analogous to yeast Hsp78 had remained elusive. The authors hypothesize that Skd3 could be the long-sought mitochondrial disaggregase, and demonstrate with an elegant series of experiments that this is indeed the case. These findings are disease-relevant, as the authors show that mutations associated with MGCA7 impair the disaggregation function of Skd3. Furthermore, the authors show that Skd3 can disaggregate preformed α-synuclein fibrils, which play a role in Parkinson’s disease and have been suggested to affect mitochondria. Given the importance of mitochondrial protein quality control, the implications of Skd3 activity may well extend more broadly to ageing and neurodegenerative diseases.



Did the authors test any other candidate mitochondrial disaggregase proteins, or was Skd3 the only obvious choice?

Are the authors aware of a correlation between Skd3 activity and ageing or neurodegenerative diseases?



  1. Nillegoda NB, Kirstein J, Szlachcic A, Berynskyy M, Stank A, Stengel F, Arnsburg K, Gao X, Scior A, Aebersold R, Guilbride DL, Wade RC, Morimoto RI, Mayer MP and Bukau B (2015) Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature 524: 247–251
  2. Gao X, Carroni M, Nussbaum-Krammer C, Mogk A, Nillegoda NB, Szlachcic A, Guilbride DL, Saibil HR, Mayer MP and Bukau B (2015) Human Hsp70 Disaggregase Reverses Parkinson’s-Linked α-Synuclein Amyloid Fibrils. Mol. Cell 59: 781–793

Tags: chaperones, mitochondria, protein disaggregation

Posted on: 13 February 2020


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Author's response

Ryan Cupo shared

Did the authors test any other candidate mitochondrial disaggregase proteins, or was Skd3 the only obvious choice?

We mainly considered Skd3 as a potential metazoan mitochondrial disaggregase for three reasons: (1) Hsp110 is required to enable potent disaggregation activity from the Hsp70/Hsp40 system, but there is no direct Hsp110 homologue in metazoan mitochondria (Shorter, 2011; Voos and Rottgers, 2002). (2) Skd3 has an HCLR clade AAA+ domain, which is involved in protein unfolding and disaggregation in other AAA+ proteins (Erzberger and Berger, 2006). Furthermore, Skd3 does not have a protease domain and lacks IGL loops that enable interaction with the ClpP protease, which suggests that any unfolding/disaggregation activity would be for resolubilization and refunctionalization rather than degradation. (3) Skd3 has a series of N-terminal ankyrin repeats which have been previously identified as an essential component of a plant protein disaggregase, cpSRP43 (Jaru-Ampornpan et al., 2010). Going forward it will be of great interest to investigate whether other protein disaggregases exist in metazoan mitochondria.

Are the authors aware of a correlation between Skd3 activity and ageing or neurodegenerative diseases?

We are not aware of association with aging or any age-associated decline in Skd3 expression. However, this area will be interesting to explore further. Due to the relatively severe nature of mutations in Skd3 and the fact that Skd3 is associated with Venetoclax (an anti-cancer drug that targets apoptotic signaling) resistance, we view Skd3 function as a strong pro-survival signal for cellular and organismal health (Chen et al., 2019; Pronicka et al., 2017).

Chen, X., Glytsou, C., Zhou, H., Narang, S., Reyna, D.E., Lopez, A., Sakellaropoulos, T., Gong, Y., Kloetgen, A., Yap, Y.S., et al. (2019). Targeting Mitochondrial Structure Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment. Cancer Discov 9, 890-909.

Erzberger, J.P., and Berger, J.M. (2006). Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu Rev Biophys Biomol Struct 35, 93-114.

Jaru-Ampornpan, P., Shen, K., Lam, V.Q., Ali, M., Doniach, S., Jia, T.Z., and Shan, S.O. (2010). ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit. Nat Struct Mol Biol 17, 696-702.

Pronicka, E., Ropacka-Lesiak, M., Trubicka, J., Pajdowska, M., Linke, M., Ostergaard, E., Saunders, C., Horsch, S., van Karnebeek, C., Yaplito-Lee, J., et al. (2017). A scoring system predicting the clinical course of CLPB defect based on the foetal and neonatal presentation of 31 patients. J Inherit Metab Dis 40, 853-860.

Shorter, J. (2011). The mammalian disaggregase machinery: Hsp110 synergizes with Hsp70 and Hsp40 to catalyze protein disaggregation and reactivation in a cell-free system. PLoS One 6, e26319.

Voos, W., and Rottgers, K. (2002). Molecular chaperones as essential mediators of mitochondrial biogenesis. Biochim Biophys Acta 1592, 51-62.

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