An Early mtUPR: Redistribution of the Nuclear Transcription Factor Rox1 to Mitochondria Protects against Intramitochondrial Proteotoxic Aggregates.

The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.

Increased mitochondrial protein import and cardiolipin remodelling upon early mtUPR.

Mitochondrial defects can cause a variety of human diseases and protective mechanisms exist to maintain mitochondrial functionality. Imbalances in mitochondrial proteostasis trigger a transcriptional program, termed mitochondrial unfolded protein response (mtUPR). However, the temporal sequence of events in mtUPR is unclear and the consequences on mitochondrial protein import are controversial. Here, we have quantitatively analyzed all main import pathways into mitochondria after different time spans of mtUPR induction. Kinetic analyses reveal that protein import into all mitochondrial subcompartments strongly increases early upon mtUPR and that this is accompanied by rapid remodelling of the mitochondrial signature lipid cardiolipin. Genetic inactivation of cardiolipin synthesis precluded stimulation of protein import and compromised cellular fitness. At late stages of mtUPR upon sustained stress, mitochondrial protein import efficiency declined. Our work clarifies the enigma of protein import upon mtUPR and identifies sequential mtUPR stages, in which an early increase in protein biogenesis to restore mitochondrial proteostasis is followed by late stages characterized by a decrease in import capacity upon prolonged stress induction.

Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei.

Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane ß-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal ß-signals. Thus in trypanosomes the highly conserved ß-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.

The versatility of the mitochondrial presequence processing machinery: cleavage, quality control and turnover.

Mitochondria play a key role in several metabolic and cell biological pathways and have attracted increasing attention due to their implication in life-span, ageing and human diseases. Mitochondrial proteases have a special role in these multiple biological functions, as they are involved in the regulation of various processes, e.g., mitochondrial protein biogenesis and quality control, mitochondrial dynamics, mitophagy and programmed cell death. The mitochondrial presequence processing machinery serves the particular purpose of maturing the majority of incoming precursor proteins by presequence cleavage, to ensure a stable mature protein by trimming of intermediate N-termini and to remove free toxic targeting peptides.

Activator and repressor functions of the Mot3 transcription factor in the osmostress response of Saccharomyces cerevisiae.

Mot3 and Rox1 are transcriptional repressors of hypoxic genes. Both factors recently have been found to be involved in the adaptive response to hyperosmotic stress, with an important function in the adjustment of ergosterol biosynthesis. Here, we determine the gene expression profile of a mot3 rox1 double mutant under acute osmostress at the genomic scale in order to identify the target genes affected by both transcription factors upon stress. Unexpectedly, we find a specific subgroup of osmostress-inducible genes to be under positive control of Mot3. These Mot3-activated stress genes also depend on the general stress activators Msn2 and Msn4. We confirm that both Mot3 and Msn4 bind directly to some promoter regions of this gene group. Furthermore, osmostress-induced binding of the Msn2 and Msn4 factors to these target promoters is severely affected by the loss of Mot3 function. The genes repressed by Mot3 and Rox1 preferentially encode proteins of the cell wall and plasma membrane. Cell conjugation was the most significantly enriched biological process which was negatively regulated by both factors and by osmotic stress. The mating response was repressed by salt stress dependent on Mot3 and Rox1 function. Taking our findings together, the Mot3 transcriptional regulator has unanticipated diverse functions in the cellular adjustment to osmotic stress, including transcriptional activation and modulation of mating efficiency.

Biosynthesis of chlorophyll c in a dinoflagellate and heterologous production in planta.

Chlorophyll c is a key photosynthetic pigment that has been used historically to classify eukaryotic algae. Despite its importance in global photosynthetic productivity, the pathway for its biosynthesis has remained elusive. Here we define the CHLOROPHYLL C SYNTHASE (CHLCS) discovered through investigation of a dinoflagellate mutant deficient in chlorophyll c. CHLCSs are proteins with chlorophyll a/b binding and 2-oxoglutarate-Fe(II) dioxygenase (2OGD) domains found in peridinin-containing dinoflagellates; other chlorophyll c-containing algae utilize enzymes with only the 2OGD domain or an unknown synthase to produce chlorophyll c. 2OGD-containing synthases across dinoflagellate, diatom, cryptophyte, and haptophyte lineages form a monophyletic group, 8 members of which were also shown to produce chlorophyll c. Chlorophyll c1 to c2 ratios in marine algae are dictated in part by chlorophyll c synthases. CHLCS heterologously expressed in planta results in the accumulation of chlorophyll c1 and c2, demonstrating a path to augment plant pigment composition with algal counterparts.

Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery.

The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.

Author's View: a nuclear transcription factor relocalizing to mitochondria rescues cells from proteotoxic aggregates.

Mitochondrial proteostasis is essential for survival, and imbalances can result in severe human diseases. We identified a novel stress response triggered upon accumulation of proteotoxic aggregates in the mitochondrial matrix. Mitochondria-to-nucleus signaling results in a transcriptional response and translocation of a nuclear transcription factor into mitochondria to maintain mitochondrial gene expression.

Dose dependent gene expression is dynamically modulated by the history, physiology and age of yeast cells.

Cells respond to external stimuli with transient gene expression changes in order to adapt to environmental alterations. However, the dose response profile of gene induction upon a given stress depends on many intrinsic and extrinsic factors. Here we show that the accurate quantification of dose dependent gene expression by live cell luciferase reporters reveals fundamental insights into stress signaling. We make the following discoveries applying this non-invasive reporter technology. (1) Signal transduction sensitivities can be compared and we apply this here to salt, oxidative and xenobiotic stress responsive transcription factors. (2) Stress signaling depends on where and how the damage is generated within the cell. Specifically we show that two ROS-generating agents, menadione and hydrogen peroxide, differ in their dependence on mitochondrial respiration. (3) Stress signaling is conditioned by the cells history. We demonstrate here that positive memory or an acquired resistance towards oxidative stress is induced dependent on the nature of the previous stress experience. (4) The metabolic state of the cell impinges on the sensitivity of stress signaling. This is shown here for the shift towards higher stress doses of the response profile for yeast cells moved from complex to synthetic medium. (5) The age of the cell conditions its transcriptional response capacity, which is demonstrated by the changes of the dose response to oxidative stress during both replicative and chronological aging. We conclude that capturing dose dependent gene expression in real time will be of invaluable help to understand stress signaling and its dynamic modulation.

In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy.

In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2ß-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.

Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast.

Cells respond to environmental stimuli by fine-tuned regulation of gene expression. Here we investigated the dose-dependent modulation of gene expression at high temporal resolution in response to nutrient and stress signals in yeast. The GAL1 activity in cell populations is modulated in a well-defined range of galactose concentrations, correlating with a dynamic change of histone remodeling and RNA polymerase II (RNAPII) association. This behavior is the result of a heterogeneous induction delay caused by decreasing inducer concentrations across the population. Chromatin remodeling appears to be the basis for the dynamic GAL1 expression, because mutants with impaired histone dynamics show severely truncated dose-response profiles. In contrast, the GRE2 promoter operates like a rapid off/on switch in response to increasing osmotic stress, with almost constant expression rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 mitogen-activated protein (MAP) kinase seem to determine the different dose-response strategies at the two promoters. Accordingly, GAL1 becomes highly sensitive and dose independent if previously stimulated because of residual Gal3 levels, whereas GRE2 expression diminishes upon repeated stimulation due to acquired stress resistance. Our analysis reveals important differences in the way dynamic signals create dose-sensitive gene expression outputs.

Deciphering dynamic dose responses of natural promoters and single cis elements upon osmotic and oxidative stress in yeast.

Fine-tuned activation of gene expression in response to stress is the result of dynamic interactions of transcription factors with specific promoter binding sites. In the study described here we used a time-resolved luciferase reporter assay in living Saccharomyces cerevisiae yeast cells to gain insights into how osmotic and oxidative stress signals modulate gene expression in a dose-sensitive manner. Specifically, the dose-response behavior of four different natural promoters (GRE2, CTT1, SOD2, and CCP1) reveals differences in their sensitivity and dynamics in response to different salt and oxidative stimuli. Characteristic dose-response profiles were also obtained for artificial promoters driven by only one type of stress-regulated consensus element, such as the cyclic AMP-responsive element, stress response element, or AP-1 site. Oxidative and osmotic stress signals activate these elements separately and with different sensitivities through different signaling molecules. Combination of stress-activated cis elements does not, in general, enhance the absolute expression levels; however, specific combinations can increase the inducibility of the promoter in response to different stress doses. Finally, we show that the stress tolerance of the cell critically modulates the dynamics of its transcriptional response in the case of oxidative stress.