Mitochondrial dysfunction is a frequent participant in common diseases and a principal suspect in aging. To combat mitochondrial dysfunction, eukaryotes have evolved a large repertoire of quality control mechanisms. One such mechanism involves the selective degradation of damaged or misfolded mitochondrial proteins by mitochondrial resident proteases, including proteases of the ATPase Associated with diverse cellular Activities (AAA+) family. The importance of the AAA+ family of mitochondrial proteases is exemplified by the fact that mutations that impair their functions cause a variety of human diseases, yet our knowledge of the cellular responses to their inactivation is limited. To address this matter, we created and characterized flies with complete or partial inactivation of the Drosophila matrix-localized AAA+ protease Lon. We found that a Lon null allele confers early larval lethality and that severely reducing Lon expression using RNAi results in shortened lifespan, locomotor impairment, and respiratory defects specific to respiratory chain complexes that contain mitochondrially encoded subunits. The respiratory chain defects of Lon knockdown (LonKD) flies appeared to result from severely reduced translation of mitochondrially encoded genes. This translational defect was not a consequence of reduced mitochondrial transcription, as evidenced by the fact that mitochondrial transcripts were elevated in abundance in LonKD flies. Rather, the translational defect of LonKD flies appeared to be derived from sequestration of mitochondrially encoded transcripts in highly dense ribonucleoparticles. The translational defect of LonKD flies was also accompanied by a substantial increase in unfolded mitochondrial proteins. Together, our findings suggest that the accumulation of unfolded mitochondrial proteins triggers a stress response that culminates in the inhibition of mitochondrial translation. Our work provides a foundation to explore the underlying molecular mechanisms.

Tim23 is an essential channel-forming subunit of the presequence translocase recruiting multiple components for assembly of the core complex, thereby regulating the protein translocation process. However, understanding of the precise interaction of subunits associating with Tim23 remains largely elusive. Our findings highlight that transmembrane helix 1 (TM1) is required for homodimerization of Tim23, while, together with TM2, it is involved in preprotein binding within the channel. Based on our evidence, we predict that the TM1 and TM2 from each dimer are involved in the formation of the central translocation pore, aided by Tim17. Furthermore, TM2 is also involved in the recruitment of Tim21 and the presequence-associated motor (PAM) subcomplex to the Tim23 channel, while the matrix-exposed loop L1 generates specificity in their association with the core complex. Strikingly, our findings indicate that the C-terminal sequence of Tim23 is dispensable for growth and functions as an inhibitor for binding of Tim21. Our model conceptually explains the cooperative function between Tam41 and Pam17 subunits, while the antagonistic activity of Tim21 predominantly determines the bound and free forms of the PAM subcomplex during import. Mitochondria are indispensable organelles required for a wide spectrum of cellular processes, including energy metabolism. The mitochondrial genome encodes only a few proteins across the kingdoms, ranging from 7 in Saccharomyces cerevisiae to 13 in humans (1, 2). Almost all the several hundreds of proteins residing in various mitochondrial compartments are encoded by the nuclear genome and synthesized on cytosolic ribosomes. Therefore, active mitochondrial function and biogenesis require efficient protein transport destined for the different subcompartments (1-9). Greater than 60% of mitochondrial proteins are targeted to the matrix compartment and are translocated through the presequence translocase (Tim23 complex), an inner membranebound multisubunit machinery whose components are highly conserved. The presequence translocase consists of central channel-forming components mainly constituted by the Tim23 protein together with Tim17, which maintains the integrity of the translocation pore (10-15), while other subunits, such as Tim50 and Tim21, which contains a single transmembrane helix with a large intermembrane space (IMS)-exposed domain, assist with presorting and guided channeling of preproteins via interaction with the translocase of outer membrane (TOM) complex (16)(17)(18)(19)(20). Besides channel-forming components, the Tim23 complex recruits a peripherally associated import motor whose functions are essential for the translocation of proteins destined for the matrix compartment (21-23). Mitochondrial heat shock protein Hsp70 (Ssc1 in yeast) forms the core of the import motor recruited for preprotein binding in the ATP-bound state to the channel via a membrane tether, Tim44 (21-24). Tim44 provides the platform and recruits two additional terminal regulatory components, J protein Pam...

The progressive accumulation of dysfunctional mitochondria is implicated in aging and in common diseases of the elderly. To oppose this occurrence, organisms employ a variety of strategies, including the selective degradation of oxidatively damaged and misfolded mitochondrial proteins. Genetic studies in yeast indicate that the ATPase Associated with diverse cellular Activities (AAA+) family of mitochondrial proteases account for a substantial fraction of this protein degradation, but their metazoan counterparts have been little studied, despite the fact that mutations in the genes encoding these proteases cause a variety of human diseases. To begin to explore the biological roles of the metazoan mitochondrial AAA+ protease family, we have created a CRISPR/Cas9 allele of the Drosophila homolog of SPG7, which encodes an inner membrane-localized AAA+ protease known as paraplegin. Drosophila SPG7 mutants exhibited shortened lifespan, progressive locomotor defects, sensitivity to chemical and environmental stress, and muscular and neuronal degeneration. Ultrastructural examination of photoreceptor neurons indicated that the neurodegenerative phenotype of SPG7 mutants initiates at the synaptic terminal. A variety of mitochondrial defects accompanied the degenerative phenotypes of SPG7 mutants, including altered axonal transport of mitochondria, accumulation of electron-dense material in the matrix of flight muscle mitochondria, reduced activities of respiratory chain complexes I and II, and severely swollen and dysmorphic mitochondria in the synaptic terminals of photoreceptors. Drosophila SPG7 mutants recapitulate key features of human diseases caused by mutations in SPG7, and thus provide a foundation for the identification of Drosophila paraplegin substrates and strategies that could be used to ameliorate the symptoms of these diseases.

Background: Although J-proteins stimulate ATPase activity of Hsp70s, the additional physiological functions are still elusive.Results: Besides its J-dependent functions, Dph4, a type III J-protein, exhibits redox activity and acts as electron carrier attributed to its iron-binding property.Conclusion: 3D structure and multiple functions of Dph4 expand the functional repertoire of the J-protein family.Significance: J-proteins have additional co-evolved specialized functions, previously not well appreciated.

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can “make or break” mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.