Deletion of HSC82 or IDS2 causes a deficiency in mitochondrial functions
We previously showed that Ids2 serves as a cochaperone of Hsc82, the major HSP90 in yeast, to maintain protein quality for cell longevity [28]. Interestingly, the growth defect of the hsc82Δ and ids2Δ cells in glycerol medium [28] was not observed in the HSP82, a paralog of HSC82, deleted cells (Additional File 1: Figures S1). We speculate that the growth defect of the hsc82Δ and ids2Δ cells may be caused by the loss of mitochondrial functions. To analyze the mitochondrial functions, we used fluorescent dyes to determine their Δψm, production of ROS, and mitochondrial mass [29, 30]. Deletion of HSC82 or IDS2 decreased the Δψm, as observed by microscopic imaging and flow cytometry analyses (Fig. 1A). The fluorescence of the ROS-sensitive probe DHE decreased significantly in stationary phase hsc82Δ and ids2Δ cells (Fig. 1B). To know whether the loss of Δψm and ROS production were due to the loss of mitochondrial function or the whole organelle, we used NAO staining to detect mitochondrial mass [31] in both fermentative (glucose) and respiratory (glycerol) growth. Under fermentative growth, the mitochondrial mass did not show a significant difference among wild-type, hsc82Δ, and ids2Δ stains. In respiratory growth, wild-type cells showed a slight increase in mitochondrial mass. However, the mitochondrial mass was decreased ~ 30% in hsc82Δ and ids2Δ cells (Fig. 1C), indicating that hsc82Δ and ids2Δ cells may have defective mitochondrial biogenesis [32]. The loss of Δψm implies that hsc82Δ and ids2Δ cells may have a defect in the respiratory chain [33]. To test this hypothesis, we tested oxygen consumption in wild-type, hsc82Δ, and ids2Δ cells. As expected, the oxygen consumption was decreased by 90% in hsc82Δ and ids2Δ cells, suggesting a severe defect in the respiratory chain (Fig. 1D). These results indicate that Hsc82 and Ids2 are essential for intact mitochondrial function.
Ids2 maintains the stability of the assembly factor of cytochrome c oxidase and ATP synthase
To understand how the loss of Ids2 causes mitochondrial dysfunction and defective cellular respiration, we screened for the non-essential proteins that physically interact with Hsc82, proteins in mitochondria, and genes required for respiratory growth from SGD (Fig. 2A and Additional File 2: Table S1). According to these three criteria, 607 Hsc82 interacting proteins were narrowed down to 20 potential clients: Aco1, Acs1, Adk1, Atp1, Atp2, Atp3, Ccs1, Cor1, Coa3, Dcs1, Fum1, Gpm1, Mir1, Ndi1, Pet9, Por1, Qcr2, Sod1, Tuf1, and Vps1. According to Gene Ontology term analysis (Additional File 3: Table S2), most of the 20 potential clients were associated with ATP metabolic process (Adk1, Atp1, Atp2, Atp3, Gpm1, Ndi1, and Qcr2) and cellular respiration (Aco1, Cor1, Fum1, Ndi1, Pet9, Qcr2, and Sod1). The potential candidates were further tested for their protein stabilities in the hsc82Δ and ids2Δ background at 30 °C and 37 °C, except for three genes not available in both TAP- and GST-tagged libraries (Dsc1, Tub1 and Vps1). Only complex IV cytochrome c oxidase assembly factor Coa3 and complex V ATP synthase subunits Atp1, Atp2, and Atp3 were markedly downregulated (Fig. 2B), while others did not exhibit a substantial decrease in the hsc82Δ and ids2Δ cells (Fig. 2C and Additional File 1: Figures S2). The mRNA levels of these potential clients were not reduced in 30 °C (Additional File 1: Figures S3). These findings suggest that Ids2 maintains the stability of cytochrome c oxidase assembly factor and ATP synthase subunits, and the alteration of the protein amounts is not regulated at the transcriptional level.
Suppression of Hsc82, Ids2, and Atp3 causes ETC damage, petite formation, and mtDNA loss
ETC generates a proton gradient across the IM by pumping protons into the IMS, which drives the synthesis of ATP via coupling with OXPHOS with ATP synthase [34]. Dramatically, all of the four potential clients were located in the ETC (Fig. 3A), and deletion of each of them showed growth defect under glycerol condition (Fig. 3B). Besides, ATP production was significantly reduced in hsc82Δ and ids2Δ cells (Fig. 3C). To further prove the deficiency in ETC, we checked the respiration-deficient petite colonies and mtDNA in atp1Δ, atp2Δ, atp3Δ, and coa3Δ cells. Strikingly, only atp3Δ, but not atp1Δ, atp2Δ, and, coa3Δ showed severe petite phenotype (Fig. 3D) and complete loss of mtDNA (Fig. 3E), as observed in the hsc82Δ and ids2Δ cells. These results demonstrate that only ATP3 deficiency causes similar mitochondrial phenotypes as the hsc82Δ and ids2Δ cells.
Atp3 is a client of Ids2
To confirm whether these four candidates are direct clients of Ids2, we analyzed their interactions with Ids2 both in vivo and in vitro. A co-immunoprecipitation assay showed that Atp1 and Atp3 interacted with Ids2 in vivo (Fig. 4A). Similar results were observed in in vitro pulldown assay where Atp1 and Atp3 displayed a mild and strong association with Ids2, respectively (Fig. 4B). To further define the major mitochondria-related client, we transformed the pRS414-ATP1 or pRS414-ATP3 plasmid into the ids2Δ cells to complement the glycerol growth defect. Only the Atp3, but not Atp1, could complement the deficiency of Ids2 (Fig. 4C). These data suggest that Atp3 may be a key client of Ids2 and the stability of other complex V candidates in ids2Δ cells might be reduced by the loss of Atp3. To test this possibility, we examined the protein stability of these candidates under the elimination of one of the candidates (Additional File 1: Figures S4A-C). Interestingly, deletion of ATP3 reduced the protein level of Atp1 and Atp2, and deletion of ATP1 or ATP2 also decreased the level of Atp3. And a co-immunoprecipitation assay observed that Atp3 co-precipitated with Ids2 and HSP90 (Additional File 1: Figures S4D), indicating that Atp3-HSP90-Ids2 may form a ternary complex. All these results suggest that Atp3 may be a major client of Ids2. To understand where Ids2 interacts with Atp3 in cells, confocal microscopic images of Ids2 were captured. Ids2-GFP distributed in cytosol, which was separated from the mitochondrial IM protein Cox4-DsRed (Fig. 4D), implying that Ids2 may interact with Atp3 in the cytosol or near the mitochondrial OM.
A middle motif in Ids2 recruits the N-terminal Atp3 to the folding system
To understand how Ids2 recruits its client Atp3, multiple truncated proteins were tested for the Ids2-Atp3 interaction. Ids2 was truncated to N-terminal (amino acid 1-92), middle (aa 92-256) and C-terminal (aa 256-469) regions, and Atp3 was truncated to ΔN (1-91 deletion), ΔM (91-225 deletion), and ΔC (225-311 deletion) forms (Additional File 1: Figures S5). To test the direct interaction, we purified the recombinant proteins of each fragment from E. coli. The pulldown results indicated that the middle domain of the Ids2 interacts with the N-terminus of Atp3 (Fig. 5A–D). According to the previous study [28], Hsc82 also interacts with the middle region of Ids2, and the Hsc82-Ids2 interaction is regulated by the phosphorylation of Ids2 S148. However, a co-immunoprecipitation assay demonstrated that the interaction between Atp3 and Ids2 S148 mutants displayed no significant difference comparing with that of the Ids2 wild-type strain (Additional File 1: Figures S6A), implying that the motif interacting with Atp3 on Ids2 is distinct from that with Hsc82. These results identify the domain requirement for Ids2 to recruit Atp3 to the folding system.
To further define the motif of Ids2 that recruits its client to the folding system, the homologs of Ids2 were aligned and three conserved motifs in the middle region of Ids2 were subjected to mutagenesis and analysis (ids2-E127A, A129G, ids2-A201G, L209A, and ids2-W219A, E225A, Additional File 1: Figures S5A). Interestingly, the ids2-A201G, L209A cells exhibited growth defects under glycerol condition (Fig. 6A) and Atp1 and Atp3 were also markedly lost in the ids2-A201G, L209A cells (Fig. 6B and Additional File 1: Figures S6B). Analysis of the secondary structure by the CFSSP program [35] identified an α-helix spanning aa 196~211 of Ids2 which covers the mutated A201 and L209 residues (Additional File 1: Figures S5A), implying that this helix may be crucial for the Ids2 cochaperone to recruit its client.
To understand how an Ids2 client is attracted to the folding system, we next examined the Atp3 sequence. Alignment and secondary structure analyses of the Atp3 homologs identified an α-helix at the N-terminal region of Atp3 spanning from aa 31 to 89 (Additional File 1: Figures S5B). We generated two mutational strains to destroy the front (atp3-41RLKS to AAAA) and the rear (atp3-66KAEK to AEAA) motifs of the α-helix, respectively Additional File 1: Figures S5B). Both atp3-41RLKS to AAAA and atp3-66KAEK to AEAA cells exhibited growth defects under glycerol condition (Fig. 6C), but Atp3-41RLKS to AAAA protein was more unstable than Atp3-66KAEK to AEAA protein (Fig. 6D). In vitro pulldown assay of Ids2 and Atp3 mutants also showed a reduction of the Ids2-Atp3 interaction (Additional File 1: Figures S6C). These results imply that the front region of the N-terminal α-helix of Atp3 may be critical in Ids2-mediated Atp3 recruitment (Fig. 6E).
Mitochondrial Yme1 and Pim1 proteases are essential for Atp3 quality control
Proteostasis highly relies on chaperones and proteases to maintain proper folding and remove unfolded proteins. Cytoplasmic proteins can be degraded by the proteasome and the vacuolar proteolysis degradation pathways [36]. On the other hand, mitochondrial sub-compartments are under surveillance of ATP-dependent proteases for unfolded and unassembled proteins [17]. To understand the protease pathways controlling the quality control of Atp3 when the HSP90/Ids2 system fails to execute its folding function, we checked whether the vacuolar protease Pep4 [37], IMS/IM protease Yme1, IM/matrix protease Yta10 [38], and matrix protease Pim1 [39] modulate the Atp3 protein level in the absence of Ids2 (Fig. 7A). Interestingly, YME1 or PIM1 deletion could rescue the Atp3 level in the ids2Δ cells (Fig. 7B and Additional File 1: Figures S7B), suggesting that Yme1 and Pim1 control the amount of Atp3. And deletion of IDS2 did not change the ratio of Atp3 inside mitochondria (Additional File 1: Figures S7C). However, only the YME1 deletion could rescue the growth defect of ids2Δ cells in the glycerol medium (Fig. 7C). These results imply that undegraded Atp3 might not be able to recover the unbalanced mitochondrial function under the loss of protease Pim1. Because Atp3 is completely undetectable in the ids2Δ cells, to study the conformational difference of Atp3 in wild-type and ids2Δ cells, we collected undegraded Atp3 from pim1Δ and ids2Δ pim1Δ strains by immunoprecipitation followed with limited Proteinase K-mediated proteolysis. Interestingly, Atp3 in the ids2Δ pim1Δ strain was more sensitive to proteolytic digestion compared with that in the pim1Δ strain (Additional File 1: Figures S7D). These results suggest that absence of Ids2 may alter the conformation of Atp3, thereby rendering it more susceptible to proteolytic digestion.
Ids2 is a mitochondria-dominant HSP90 cochaperone induced in the glycerol medium
Given that Ids2 is essential for mitochondria function, we asked whether Ids2 is a mitochondria-dominant HSP90 cochaperone induced under the requirement of oxidative respiration. A major cytoplasmic HSP90 cochaperone is Aha1, which also binds to the middle domain of HSP90 [40] and promotes Hsc82 ATPase activity as Ids2 [28]. We, therefore, compared the growth of wild-type, hsc82Δ, ids2Δ, and aha1Δ cells in the glycerol medium. Interestingly, aha1Δ cells did not exhibit a growth defect in glycerol (Additional File 1: Figures S8A). Atp3 was also stably maintained in the aha1Δ cells (Additional File 1: Figures S8B). In contrast, Ids2 was highly expressed in the glycerol medium (Additional File 1: Figures S8C). These results suggest that Ids2 is more important for mitochondrial function than cytoplasmic HSP90 cochaperone Aha1.