NEK10 interactome and depletion reveal new roles in mitochondria.

BACKGROUND: Members of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions. METHODS: HEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established. RESULTS: Here, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content. CONCLUSIONS: Taken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.

Cardiolipin is a key determinant for mtDNA stability and segregation during mitochondrial stress.

Mitochondria play a key role in adaptation during stressing situations. Cardiolipin, the main anionic phospholipid in mitochondrial membranes, is expected to be a determinant in this adaptive mechanism since it modulates the activity of most membrane proteins. Here, we used Saccharomyces cerevisiae subjected to conditions that affect mitochondrial metabolism as a model to determine the possible role of cardiolipin in stress adaptation. Interestingly, we found that thermal stress promotes a 30% increase in the cardiolipin content and modifies the physical state of mitochondrial membranes. These changes have effects on mtDNA stability, adapting cells to thermal stress. Conversely, this effect is cardiolipin-dependent since a cardiolipin synthase-null mutant strain is unable to adapt to thermal stress as observed by a 60% increase of cells lacking mtDNA (ρ0). Interestingly, we found that the loss of cardiolipin specifically affects the segregation of mtDNA to daughter cells, leading to a respiratory deficient phenotype after replication. We also provide evidence that mtDNA physically interacts with cardiolipin both in S. cerevisiae and in mammalian mitochondria. Overall, our results demonstrate that the mitochondrial lipid cardiolipin is a key determinant in the maintenance of mtDNA stability and segregation.

DivIVA-mediated polar localization of ComN, a posttranscriptional regulator of Bacillus subtilis.

ComN (YrzD) is a small, 98-amino-acid protein recently shown to be involved in the posttranscriptional control of the late competence comE operon in Bacillus subtilis. We show here that ComN localizes to the division site and cell poles in a DivIVA-dependent fashion. Yeast two-hybrid and glutathione S-transferase pulldown experiments showed that ComN interacts directly with DivIVA. ComN is not essential for the polar assembly of the core competence DNA uptake machinery. Nevertheless, polar localization of ComN should play some role in competence acquisition because delocalization of ComN leads to a small reduction in competence efficiency. We found that ComN promotes the accumulation of its target comE mRNA to septal and polar sites. Thus, we speculate that localized translation of ComE proteins may be required for efficient competence development. Our results underscore the versatility of DivIVA as a promoter of the differentiation of bacterial poles and demonstrate that the repertoire of polarly localized molecules in B. subtilis is broad, including a regulator of gene expression and its target mRNA. Moreover, our findings suggest that mRNA localization may play a role in the subcellular organization of bacteria.