Biology subjects

Herrmann, J. M.

Publications and source records attributed to Herrmann, J. M..

16 recordsLinked to original sources

bioRxiv2026

Whole genome duplication uncouples cell volume scaling from mitochondrial homeostasis

Whole genome duplication, or polyploidy, increases cell size and nuclear volume, yet how cells maintain cytonuclear balance and metabolic fitness following ploidy expansion remains unclear. Using isogenic series of Saccharomyces cerevisiae strains ranging from haploid (1N) to tetraploid (4N), and isogenic human diploid and tetraploid cell lines, we investigated the impact of polyploidy on mitochondrial homeostasis and metabolic adaptation. Quantitative proteomics uncovered a sub-linear scaling of mitochondrial proteins with increasing ploidy, with pronounced depletion of respiratory chain components, mitochondrial DNA maintenance factors, and redox homeostasis proteins. These changes were highly conserved, indicating a conserved consequence of genome expansion. Impaired mitochondrial scaling reduced respiratory growth, membrane potential and oxygen consumption, and elevated oxidative stress. In budding yeast, the mitochondrial dysfunction triggered reactive oxygen species (ROS) accumulation and an unconventional activation of stress-responsive signaling pathways, including the Hog1 and cell integrity MAPK networks, which converged on Rtg2-dependent retrograde signaling to promote mitochondrial metabolism despite chronic dysfunction. Our findings identify defective mitochondrial scaling as a key consequence of polyploidy and establish ROS- and stress signaling-dependent retrograde response as a key adaptive mechanism linking genome duplication to mitochondrial homeostasis and cellular fitness.

cell biology↗

bioRxiv2026

Presequences of non-imported mitochondrial proteins serve as quality control elements in the cytosol

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with presequences which serve as targeting signals for the mitochondrial matrix, where they are cleaved by the mitochondrial processing peptidase (MPP). In this study, we comprehensively elucidated the role of the presequence and the mature part of mitochondrial precursors in the cytosol, by use of a cytosol-targeted MPP which prematurely processed mitochondrial precursors. Over time, cytoMPP resulted in mitochondrial depletion. However, the cellular response to cytoMPP was surprisingly different to that observed for other models of mitochondrial import inhibition. Cytosolic maturation rendered many proteins stable in the cytosol, indicating that their mature parts lack ubiquitination signals. Accordingly, cytoMPP did not induce the upregulation of the proteasome, which normally is a hallmark of mitochondrial dysfunction. Instead, cytoMPP elicited a heat shock response and impaired the sequestration of precursors in the cytosol. Our observations demonstrate that mitochondrial presequences are more than just address labels. Rather, they play an important role in quality control and orchestrate the cellular response to defects in mitochondrial protein import.

biochemistry↗

bioRxiv2026

The mitochondrial cohibitin complex facilitates the biogenesis of inner membrane proteins

The inner membrane of mitochondria contains many membrane-embedded carrier proteins of the SLC25 family to facilitate the exchange of metabolites between the cytosol and mitochondria. These carriers use a specific import route for their biogenesis that relies on the TIM22 complex as an inner membrane translocase. The molecular details of carrier biogenesis are not well understood. Using an improved, desthiobiotin-based proximity labeling approach called Destni in living yeast cells, we identified the mitochondrial protein Aim11 as an interactor of newly imported carrier proteins. Aim11 forms a 160 kDa complex together with Iai11, Gep7 and Mtc1 in the mitochondrial inner membrane that we named the comrade-of-prohibitin (cohibitin) complex owing to its genetic interaction with prohibitins. Deletion of Aim11 impairs the import of carrier proteins into the inner membrane and renders cells hypersensitive to carrier overexpression. Our data suggest that the cohibitin complex plays a quality control function that supports the TIM22-mediated insertion of carrier proteins into the inner membrane of mitochondria. SummaryKizmaz et al. identified Aim11 as a novel quality control factor that facilitates the insertion of carrier proteins into the inner membrane of mitochondria. Aim11 is part of the membrane-embedded cohibitin complex which cooperates with prohibitins in inner membrane protein biogenesis.

biochemistry↗

bioRxiv2025

Succinate and its carrier Sfc1 mediate metabolic control of mitochondrial protein import by the TIM23 translocase

Tim23 is an essential component of the mitochondrial inner membrane translocase and Sfc1 is a carrier that exchanges succinate for fumarate across that membrane. Sfc1 and succinic acid availability regulate dual targeting of fumarase and aconitase by facilitating mitochondrial import of their newly synthesized precursors, as shown by pulse-chase experiments. Here we show that Sfc1 directly interacts with the Tim23 and succinate affects this interaction, which in turn affects mitochondrial protein import. Physical interaction between Tim23 and Sfc1 was proven by co immunoprecipitation, Bimolecular Fluorescence Complementation (BiFC) and Biotin-based proximity labeling (TurboID). Proximity labeling-informed mutagenesis allowed us to dissect the carrier activity of Sfc1 from its function as a TIM23 regulator. We performed Rosetta-MP docking of Sfc1 and Tim23 to envisage the interface. Thus, our findings show that metabolites can regulate mitochondrial import and adjust the segregation of key metabolic enzymes between the cytosol and mitochondria.

cell biology↗

bioRxiv2024

A priority code in presequences: mitochondrial targeting signals assign specific import characteristics to precursor proteins

The biogenesis of mitochondria relies on the import of hundreds of different precursor proteins from the cytosol. Most of these proteins are synthesized with N-terminal presequences which serve as mitochondrial targeting signals. Presequences consistently form amphipathic helices, but they considerably differ in respect to their primary structure and length. Here we show that presequences can be classified into seven different groups based on their specific features. Using a test set of different presequences, we observed that group A presequences endow precursor proteins with improved in vitro import characteristics. We developed IQ-Compete (for Import and de-Quenching Competition assay), a novel assay based on fluorescence de-quenching, to monitor the import efficiencies of mitochondrial precursors in vivo. With this assay, we confirmed the increased import competence of group A presequences. Using mass spectrometry, we found that the presequence of the group A protein Oxa1 specifically recruits the tetratricopeptide repeat (TPR) containing protein TOMM34 to the cytosolic precursor protein. TOMM34 apparently serves as a presequence-specific targeting factor which increases the import efficiency of a specific subset of mitochondrial precursor proteins. Our results suggest that presequences contain a protein-specific priority code that encrypts the targeting mechanism of individual mitochondrial precursor proteins.

biochemistry↗

bioRxiv2023

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER encounter structure (ERMES) and Tom70 are part of two parallel and partially redundant ER-to-mitochondria transfer routes. When ER-to-mitochondria transfer is prevented, many mitochondrial precursor proteins associate with ER membranes, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

biochemistry↗

bioRxiv2022

Mitochondrial dysfunction rapidly modulates the abundance and thermal stability of cellular proteins

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger the mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling (ppTPP). This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labeling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Strikingly, different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of the functional modules that are relevant for mitoprotein-induced stress. Thus, our new ppTPP approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of protein abundance and protein stability.

biochemistry↗

bioRxiv2022

MitoStores: Chaperone-controlled protein granules store mitochondrial precursors in the cytosol

Hundreds of mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational reaction. The early processes associated with mitochondrial protein targeting remain poorly understood. Here we show that in bakers yeast, the cytosol has the capacity to transiently store matrix-destined precursors in dedicated deposits which we named MitoStores. MitoStores are strongly enhanced when protein import into mitochondria is competitively inhibited by a clogging of mitochondrial import sites, but also formed under physiological conditions when cells grow on non-fermentable carbon sources. MitoStores are enriched for a specific subset of nuclear encoded mitochondrial proteins, in particular those containing N-terminal mitochondrial targeting sequences. MitoStore formation is controlled by the heat shock proteins Hsp42 and Hsp104, potentially to suppress the toxic potential of accumulating mitochondrial precursor proteins. Thus, the cytosolic protein quality control system plays an active role during early stages in mitochondrial protein targeting by the coordinated and localized sequestration of mitochondrial precursor proteins. SummaryThe yeast cytosol can deposit precursors of mitochondrial proteins in specific granules called MitoStores. MitoStores are controlled by the cytosolic chaperone system, in particular by Hsp42 and Hsp104. MitoStore formation suppresses the toxicity arising from non-imported mitochondrial precursor proteins.

biochemistry↗

bioRxiv2022

The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import by enhancing assembly of the TOM complex

Mitochondria are essential organelles that play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenesis enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and stabilizes its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in a compromised protein import into mitochondria and a subsequent accumulation of mitochondrial precursor proteins in the cytosol. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery in order to improve mitochondrial protein import during phases of metabolic transition.

biochemistry↗

bioRxiv2021

The ER transmembrane complex (EMC) can functionally replace the Oxa1 insertase in mitochondria

Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): The guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, that are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2-Get1 and Emc6-Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6-Emc3 fusion was able to complement an{Delta} oxa1 mutant and restored its respiratory competence. The Emc6-Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2 as well as of nuclear encoded inner membrane proteins though was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.

biochemistry↗

bioRxiv2021

The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins

Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPRER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The levels of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPRER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that cant be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

cell biology↗

bioRxiv2021

Mitochondrial ribosomal proteins developed unconventional mitochondrial targeting signals due to structural constraints

Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Available proteomic and structural data as well as computational predictions indicate that up to 25% of MRPs do not have a conventional N-terminal mitochondrial targeting signal (MTS). We characterized a set of 15 yeast MRPs in vivo and showed that 30% of them use internal mitochondrial targeting signals. We isolated a novel internal targeting signal from the conserved MRP Mrp17 (bS6). The Mrp17 targeting signal shares some properties as well as import components with conventional MTS-containing proteins but is not reliably predicted indicating that mitochondrial protein targeting is more versatile than expected. We hypothesize that internal targeting signals arose in MRPs when the N-terminus extension was constrained by ribosome assembly interfaces.

cell biology↗

bioRxiv2021

Increased levels of the mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient material and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but the causalities remained unclear. We used yeast as model system to analyze the relevance of mitochondrial processes for the behavior of an aggregation-prone polyQ protein derived from human huntingtin. Induction of Q97-GFP rapidly leads to insoluble cytosolic aggregates and cell death. Although this aggregation impairs mitochondrial respiration only slightly, it interferes with efficient import of mitochondrial precursor proteins. Mutants in the import component Mia40 are hypersensitive to Q97-GFP. Even more surprisingly, Mia40 overexpression strongly suppresses the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the posttranslational import into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Owing to its rate-limiting role for mitochondrial protein import, Mia40 acts as a regulatory component in this competition. This role of Mia40 as dynamic regulator in mitochondrial biogenesis can apparently be exploited to stabilize cytosolic proteostasis. (174/175 words)

biochemistry↗

bioRxiv2020

The mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress

Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high content screens, we revisited the question of Tom70 function and considerably expanded the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, in particular of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondria-specifying targeting receptor.

cell biology↗

bioRxiv2020

More than just a ticket canceller: The mitochondrial processing peptidase matures complex precursor proteins at internal cleavage sites

Most mitochondrial proteins are synthesized in the cytosol as precursors that carry N-terminal presequences. After import into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase MPP, giving rise to shorter mature proteins. Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into the mitochondrial matrix and subsequently separated into two distinct enzymes that function in arginine biogenesis. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor both at its N-terminus and at an internal site between the Arg5 and Arg6 parts. The peculiar organization and biogenesis of Arg5,6 is conserved across fungi and might preserve the mode of co-translational subunit association of the arginine biosynthesis complex of the polycistronic arginine operon in prokaryotic mitochondrial ancestors. Putative MPP cleavage sites are also present at the junctions in other mitochondrial fusion proteins from fungi, plants and animals. Our data suggest that, in addition to its role as “ticket canceller” for the removal of presequences, MPP exhibits a second, widely conserved activity as internal processing peptidase for complex mitochondrial precursor proteins.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

Multiple acyl-CoA thioesterases occupy distinct functional niches within the mitochondrial matrix

Acyl-CoA thioesterases (Acots) hydrolyze fatty acyl-CoA esters. Acots in the mitochondrial matrix are poised to mitigate {beta}-oxidation overload that may contribute to lipotoxicity. Several Acots associate with mitochondria, but whether they all localize to the matrix, and are redundant or have different roles is unresolved. We compared mitochondrial Acots (Acot2, 7, 9, and 13) in terms of suborganellar localization, activity, expression and regulation, in mitochondria from multiple mouse tissues and from a new model of Acot2 depletion. Acot7, 9 and 13 localized to the matrix, joining Acot2 that was previously shown to localize there. Mitochondria from heart, skeletal muscle, brown adipose tissue and kidney robustly expressed Acot2, 9 and 13, though Acot9 was substantially higher in brown adipose tissue and kidney mitochondria, as was activity for C4:0-CoA, a unique substrate of Acot9. In all these tissues, Acot2 accounted for [~]half of the thioesterase activity for C14-CoA and C16:0-CoA. In contrast, liver mitochondria from fed and fasted mice expressed little Acot activity, and this activity was confined to long-chain CoAs, and due mainly to Acot7 and Acot13 activity. Matrix Acots occupied different functional niches, based on substrate specificity (Acot9 vs. Acot2 and 13) and strong CoA inhibition (Acot7, 9, 13 but not Acot2). Interpreting these results in the context of {beta}-oxidation, CoA inhibition would prevent Acot-mediated suppression of {beta}-oxidation while providing for an Acot-mediated release valve when CoA is limiting. This release valve would operate across a wide range of acyl-CoA chain lengths. In contrast, CoA-insensitive Acot2 could provide a constitutive syphon for long-chain fatty acyl-CoAs. These results reveal how the family of matrix Acots can help to mitigate {beta}-oxidation overload and prevent a CoA limitation.

biochemistry↗