M BoC | ARTICLE Stoichiometric expression of mtHsp40 and mtHsp70 modulates mitochondrial morphology and cristae structure via Opa1L cleavage Byoungchun Leea, Younghee Ahnb, Sung-Myung Kanga, Youngjin Parkc, You-Jin Jeond, Jong M. Rhob, and Sung-Woo Kimb aSouthern Alberta Cancer Research Institute, Department of Biochemistry and Molecular Biology, and bDepartment of Pediatrics and Clinical Neurosciences, Alberta Children’s Hospital Research Institute for Child and Maternal Health, University of Calgary, Calgary, AB T2N 4N1, Canada; cCollege of Medicine, University of Dong-A, Dong-A Medical Center, Busan 602-714, Republic of Korea; dDepartment of Marine Life Science, Jeju National University, Jeju 690-756, Republic of Korea ABSTRACT Deregulation of mitochondrial heat-shock protein 40 (mtHsp40) and dysfunction Monitoring Editor of mtHsp70 are associated with mitochondrial fragmentation, suggesting that mtHsp40 and Thomas D. Fox mtHsp70 may play roles in modulating mitochondrial morphology. However, the mechanism Cornell University of mitochondrial fragmentation induced by mtHsp40 deregulation and mtHsp70 dysfunction Received: Feb 25, 2014 remains unclear. In addition, the functional link between mitochondrial morphology change Revised: Mar 19, 2015 upon deregulated mtHsp40/mtHsp70 and mitochondrial function has been unexplored. Our Accepted: Apr 10, 2015 coimmunoprecipitation and protein aggregation analysis showed that both overexpression and depletion of mtHsp40 accumulated aggregated proteins in fragmented mitochondria. Moreover, mtHsp70 loss and expression of a mtHsp70 mutant lacking the client-binding do- main caused mitochondrial fragmentation. Together the data suggest that the molecular ra- tio of mtHsp40 to mtHsp70 is important for their chaperone function and mitochondrial morphology. Whereas mitochondrial translocation of Drp1 was not altered, optic atrophy 1 (Opa1) short isoform accumulated in fragmented mitochondria, suggesting that mitochon- drial fragmentation in this study results from aberration of mitochondrial inner membrane fusion. Finally, we found that fragmented mitochondria were defective in cristae develop- ment, OXPHOS, and ATP production. Taken together, our data suggest that impaired stoichi- ometry between mtHsp40 and mtHsp70 promotes Opa1L cleavage, leading to cristae open- ing, decreased OXPHOS, and triggering of mitochondrial fragmentation after reduction in their chaperone function. INTRODUCTION Opposing mitochondrial fusion and fission are key events regulating functional pool of mitochondria (Collins et al., 2002; Cereghetti mitochondrial morphology and play critical roles in maintaining a et al., 2008; Gomes et al., 2011). During promotion of mitochondrial fission, the cytosolic GTPase dynamin-related protein 1 (Drp1) is re- cruited independently to the mitochondrial outer membrane by an This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E14-02-0762) on April 22, 2015. array of integral outer membrane proteins—mitochondrial fission Address correspondence to: Sung-Woo Kim ([email protected]). protein 1 (Fis1), mitochondrial fission factor (Mff), and mitochondrial Abbreviations used: DOX, doxycycline; FCCP, carbonyl cyanide-4-(trifluorometh- dynamics proteins of 49 and 51 kDa (MiD49 and MiD51; Yoon et al., oxy)phenylhydrazone; MKT-077, 1-ethyl-2-[[3-ethyl-5-(3-methyl-2(3H)-benzothi- 2003; Otera et al., 2010; Zhao et al., 2011; Loson et al., 2013). More- azolylidene)-4-oxo-2-thiazolidinylidene]methyl]-pyridinium chloride; NP40, Noni- det P-40; RNAi, RNA interference; ROT, rotenone. over, mitochondria sense mitochondrial stresses such as membrane © 2015 Lee et al. This article is distributed by The American Society for Cell Biol- depolarization and apoptotic cell death, which shifts mitochondrial ogy under license from the author(s). Two months after publication it is available morphology to fragmented punctae (Cereghetti et al., 2008; Suen to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). et al., 2008). Mitochondrial fusion depends on two outer mem- “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of brane–bound GTPases—mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2)— the Cell®” are registered trademarks of The American Society for Cell Biology. and one inner membrane–bound GTPase, optic atrophy 1 (Opa1; 2156 | B. Lee et al. Molecular Biology of the Cell Chen et al., 2003; Cipolat et al., 2004). Of interest, Opa1 has several (0–100 ng/ml) of DOX. DOX at 5 ng/ml induced mtHsp40L 33-fold isotypes that are soluble in the intermembrane space or bound to higher than control, and ∼80% of cells had severely fragmented mi- inner membrane and coordinate cristae junctions, promoting cristae tochondria (Figure 1, D and E). DOX doses >5 ng/ml had no addi- density and inner membrane fusion (Frezza et al., 2006; Loson et al., tional effect on mitochondrial morphology. 2013). It has been reported that genetic depletion of Mfn1/2 and Mitochondrial fragmentation is reversibly induced in a response accumulation of Opa1 short isoform (Opa1S) after the cleavage of to various stresses such as loss of membrane potential (Legros et al., Opa1L promote mitochondrial fragmentation (Guillery et al., 2008; 2002; Jendrach et al., 2008). Thus we tested whether mitochondrial Gegg et al., 2010). morphology change here is also reversible according to mtHsp40 The mitochondrial heat-shock proteins mtHsp40 and mtHsp70 level. Of interest, most mitochondria reverted to tubules (Figure 1G are classified in the Hsp40 and Hsp70 families, respectively. Mecha- and Supplemental Figure S1) after mtHsp40L levels returned to con- nistically, mtHsp40 and mtHsp70 cooperate to maintain mitochon- trol level (Figure 1F). These data suggest that the aberrant levels of drial proteostasis by supporting protein (re)folding (Hartl et al., mtHsp40 reversibly caused mitochondrial fragmentation. 2011). Both client-bound and free forms of mtHsp40 interact with the ATPase domain of mtHsp70 (Kampinga and Craig, 2010). In vitro mtHsp70 client-binding activity is decreased in findings demonstrated that elevated mtHsp40 protein without con- mtHsp40-overexpressing cells comitant increase of mtHsp70 protein inhibited mtHsp70 activity of Both isoforms of mtHsp40—mtHsp40L and mtHsp40S—physically protein deaggregation (Hageman et al., 2011; Iosefson et al., 2012), interact with mtHsp70 to promote mitochondrial protein stability indicating that the stoichiometry of mtHsp40–mtHsp70 is critical for (Ahn et al., 2010; Iosefson et al., 2012). To test the effect of mtHsp40L their chaperone activity. Both mtHsp40L and mtHsp40S, two splice expression on mtHsp70 interaction with endogenous mtHsp40S, we variants of mtHsp40, bind mtHsp70 to mediate (re)folding of nascent isolated mitochondria from control and mtHsp40L-overexpressing polypeptides in vitro and in vivo (Ahn et al., 2010; Iosefson et al., cells, followed by coimmunoprecipitation using anti-mtHsp70 anti- 2012), and overexpression of both forms promotes apoptosis (Kim bodies. We found that the level of mtHsp40S pulled down by anti- et al., 2004). One study showed that loss-of-function mutation of mtHsp70 antibodies in mtHsp40L-overexpressing cells was substan- mtHsp70 in Parkinson disease patients increased mitochondrial pro- tially lower than in control (Figure 2A), suggesting that mtHsp70 tein instability, leading to mitochondrial fragmentation (Burbulla interaction with endogenous mtHsp40S was inhibited in fragmented et al., 2010). In addition, our recent study showed that deregulation mitochondria after ectopic expression of mtHsp40L. To confirm this of mtHsp40 resulted in mitochondrial fragmentation in a way that observation, we repeated this experiment from cells treated with a depended on Drp1 (Lee et al., 2012), suggesting that mtHsp40 may range of DOX concentrations. We found that mtHsp70 interaction play important roles together with mtHsp70 in modulating mito- with endogenous mtHsp40S was negatively affected with ≥5 ng/ml chondrial morphology. However, little is known about the link be- DOX (Figure 2B). Given that mtHsp70 requires mtHsp40 in order to tween mitochondrial morphology and the chaperone function of bind client proteins (Kampinga and Craig, 2010), we thus proposed mtHsp40–mtHsp70. Although it has been proposed that Drp1 is re- that mtHsp40 higher than a threshold can inhibit mtHsp70 interac- quired for mitochondrial fragmentation, it is unclear how the deregu- tion with client proteins, leading to reduction of protein (re)folding. lated ratio of mtHsp40–mtHsp70 affects mitochondrial morphology. To test this hypothesis, we performed a protein aggregation assay. Finally, the relationship between mitochondrial morphology and Consistent with previous in vitro experiments (Iosefson et al., 2012), function has been unexplored. We therefore set out to investigate overexpression of mtHsp40L showed the tendency of elevated pro- how mitochondria become fragmented and which mitochondrial tein aggregation in fragmented mitochondria in the presence of function is altered by finely altering the ratio of mtHsp40–mtHsp70. NP-40 detergent (Figure 2C). Taken together, these data suggest Our results show that the molecular ratio of mtHsp40–mtHsp70 de- that overexpression of mtHsp40 inhibits the chaperone function of termines mitochondrial morphology, cristae formation, and oxida- mtHsp70, leading to fragmented mitochondria.