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To whom correspondence should be addressed: Dept. of Biochemistry, Indian Institute of Science, Biological Sciences Bldg., Bangalore 560012, Karnataka, India. Tel.: 91-080-22932821; Fax: 91-080-23600814
* This work was supported by Swarnajayanti Fellowship, Department of Science and Technology (DST), India (to P. D. S), Council of Scientific and Industrial Research Fellowship (to D. S), and Department of Science and Technology INSPIRE Fellowship (to S. S). The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Figs. S1–S4. 1 Present address: Dept. of Zoology, Institute of Science, Banaras Hindu University, Uttar Pradesh, Varanasi 221005, India.
Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel.
). More than 98% of the human mitochondrial protein pool is nuclear-encoded and transported into the mitochondria through highly complex import machinery spanning the outer membrane, inner membrane, and the intermembrane space (
). The TIM23 complex comprises of two components: membrane-embedded Tim23 core channel and the motor component. Although the initial membrane potential dependent translocation is mediated by the Tim23-Tim17 channel, it is the overall activity of the import motor, which determines the translocation process (
In yeast, mitochondrial import motor consists of a single J-protein, Pam18, which stimulates yeast mitochondrial Hsp70 (Ssc1) ATPase activity. Pam18 forms a heterodimeric subcomplex with Pam16, a J-like protein that inhibits its ATPase stimulatory activity and thereby exerts a regulatory role on the overall activity of import motor and the transport process (
). Homologs of Pam16 and Pam18-like proteins are found across all eukaryotic species. The ortholog of Pam16, identified in humans as Magmas, is essential for biogenesis of mitochondria and also plays an additional role in regulation of reactive oxygen species in humans (
). A recent observation showed Magmas to function as a ROS (reactive oxygen species) regulator where it maintains the cellular redox state under stress conditions by promoting efficient oxidative phosphorylation (OXPHOS) activity and enhanced reactive oxygen species scavenging (
). Both JC19 and JC15 are related to Pam18 in several aspects. Both have a significant sequence and structural similarity with its yeast ortholog. The C-terminal region of JC15 and JC19 consists of three helices similar to that of the Pam18 J-domain with a tightly packed canonical HPD motif between its helix II and helix III. Both localize into the mitochondria and are associated with the inner mitochondrial membrane via a single transmembrane helix with its J-domain facing the matrix side.
Interestingly, JC15 was first identified as a protein whose hypermethylation-mediated transcriptional silencing resulted in increased resistance to chemotherapeutic agents in ovarian carcinoma cells (
). The CpG island at the first exon of the DNAJC15 gene was found to be methylated, and the number of methylated CpG sites in the island governs the extent of JC15 expression in the cancer cells and in normal tissues (
). JC15 expression was found to decrease cellular survivability under xenobiotic stress conditions by modulating the opening of mitochondrial permeability transition pore complex through its association with cyclophilin D (
). Hence, it was suggested that loss of expression of JC15 contributes to the malignant phenotype by conferring resistance to various anti-cancer drugs by modulating the chemotherapeutic response of the cancer cells and served as a molecular marker for the response to chemotherapy (
Although much study has been done on the relationship of the J-proteins and cellular phenotype, no comparative information on the specificities of J-proteins is available. Here we show that unlike JC19, JC15 could complement growth of yeast cells deleted for PAM18 and shows characteristics that are functionally similar to Pam18. In addition, JC15 was found to possess a conserved function of eliciting a chemosensitive response in yeast cells, which is indicative of its dual function in eukaryotic cell. We characterized the behavior of both the J-proteins in human cells, and our results indicate that the differential function of the two J-proteins is independent of their subcomplex formation with Magmas.
In the present study we comparatively assessed the functional specificities of both the J-proteins in human mitochondria and showed JC15 to be a bifunctional protein, having roles both in protein transport and chemosensitivity. JC15 was documented as a protein whose epigenetic silencing regulates the response to chemotherapeutic drug treatment and in normal cells of epithelial origin (
). However, both JC19 and JC15 form a stable heterodimer with that of Magmas through their J-domain with equal affinity, which like other membrane-associated J-proteins, is the minimal region required for the interaction.
The protein translocation activity in mitochondria requires efficient chaperoning activity of mtHsp70 to vectorially pull the incoming polypeptide into the matrix (
). The protein translocation function, being an ATP-dependent process, requires optimum ATPase activity of mtHsp70, which is stimulated by the J-protein counterpart Pam18 in the case of yeast. Loss of Pam18 function has been shown to be lethal for the yeast cell (
). The ability of JC15 to complement Pam18 function in yeast by rescuing the viability of Δpam18 cells and efficiently importing the precursor proteins is probably due to their higher sequence complementarity and similar domain organization, thus suggesting that JC15 was evolutionary more closely related to Pam18 than JC19 which lacks the ability to rescue Δpam18 cells (
). This is also supported by the fact that JC15 could stably interact with Pam16, form a part of TIM23 complex, and efficiently stimulate mtHsp70 ATPase activity. Loss of function mutations in the JC15 J-domain compromises its association with the translocon due to disruption of the subcomplex formation with Pam16 and causes dysregulation of import motor activity. This results in severe growth defects and translocation phenotypes, thereby affecting mitochondrial function and biogenesis.
In contrast to yeast, where there is a single J-protein Pam18 that is associated with the TIM23 complex (
), humans present a unique case of having two J-proteins, namely JC19 and JC15, as a part of the inner membrane translocation machineries (Fig. 9). Both the J-protein paralogs form two mutually distinct and functionally independent subcomplexes with their common J-like protein counterpart Magmas (Fig. 9). However, the majority of the protein transport processes are regulated by JC19, thereby playing a determining role in human mitochondria biogenesis, in contrast to JC15 which has a variable tissue-specific expression (
). The presence of two J-proteins involved in functional heterodimer with Magmas at the import channel assumes importance due to the fact that both JC19 and JC15 are associated with contrasting cellular phenotype (
). A heterozygous mutation at the C-terminal region of JC19 leads to the formation of shorter protein with a truncated J-domain. This results in a debilitating disorder dilated cardiomyopathy and ataxia syndrome (DCM). DCM patients show profound neurodegeneration, skeletal muscle wastage, cardiac disorders leading to ataxia, and early age mortality (
). On the other hand, apart from its supportive role in protein transport, JC15 is suggested to modulate the organization and activity of electron chain complexes, a function also attributed to Magmas, thus suggesting a possible role of JC15·Magmas subcomplex in regulation of respiratory activity (
). Studies in cultured cells indicate stress-dependent association of JC15 with cyclophilin D in response to cisplatin treatment, which is responsible for recruitment of cyclophilin D to the mitochondrial transition pore complex, resulting in channel opening and induction of cell death (
). We found the chemomodulatory function of JC15 to be conserved across species, as yeast cells expressing the protein showed enhanced sensitivity to the drug. JC15 in addition to its complementation of the essential protein import function conferred increased drug sensitivity to yeast cells, highlighting its multifunctional role in the cell. The level of sensitivity was, however, proportional to the amount of protein expressed. Our results also highlight that the J-domain of JC15 plays a critical role in the overall process leading to development of the chemosensitive response.
The mitochondrial inner membrane J-proteins are thought to function in association with their corresponding J-like protein counterparts (
). The contrasting functions of both the J-proteins paralogs, however, do not reflect on their properties at the translocase channel (Fig. 9). Both JC19 and JC15 have equal affinity of interaction with Magmas, stimulate human mtHsp70's activity to similar levels, and along with Magmas form higher order oligomeric structures unique to the mammalian system. Although a reduced level of JC15 is dispensable for cell survival and protein translocation, depletion of JC19 results in increased cell mortality, protein import defects, and decreased mitochondrial function, thus highlighting the inability to JC15 to complement JC19 function in vivo. This is consistent with clinical facts that DCM patients have a higher mortality rate with reduced life span, and the absence of JC15 expression is well tolerated by the cell as observed in normal fibroblasts and proliferating chemorefractory cancer cells (
). This indicates that the differential function of the two J-proteins in determining the cellular phenotype is independent of their individual association with the translocation channel and might be governed by the local primary sequence or secondary molecules, which are the focus of future investigations.
D. S. and P. D. S. conceived the study and designed the experiments. D. S. performed the majority of the experiments. S. S. conducted experiments related to the growth phenotypes. All authors analyzed the data and wrote the manuscript.
We thank Prof. Elizabeth Craig for providing yeast strains and yeast-specific protein antibodies. We acknowledge the Bio-Rad Haifa facility, Israel, for performing surface plasmon resonance experiments. We thank the Flow Cytometry facility of Indian Institute of Science, Bangalore, for flow cytometry experiments.