Biology subjects

Raeschle, M.

Publications and source records attributed to Raeschle, M..

17 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

Analysis of stress-induced surfaceome remodeling reveals surface accumulation of the cation-independent mannose-6-phosphate receptor (CI-M6PR)

To ensure their survival in the face of stressors, cells have evolved stress response programs. While several transcriptional stress responses have been elucidated, little is known about the impact of stressors on membrane transport and the protein composition of the cell surface. Yet, the dynamic remodeling of the surfaceome by processes such as endocytosis is likely central for the adaptation to stress as it shapes cellular responses by influencing ion uptake and numerous signaling cascades. Indeed, we show that different stressors decrease endocytosis, thereby facilitating cellular adaptation. Using quantitative mass spectrometry, we delineate stress-specific surfaceome alterations in response to osmotic, oxidative and heat stress. Among other adaptive changes, we uncover that osmotic stress leads to a striking surface accumulation of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Mechanistically, we demonstrate that osmotic stress decreases the endocytosis of CI-M6PR while upregulating its lysosomal exocytosis. These results suggest that CI-M6PR might play an important role in the cellular resilience against osmotic stress.

cell biology↗

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

Pex30-dependent membrane contacts sites maintain ER lipid homeostasis

In eukaryotic cells, communication between organelles and the coordination of their activities depend on membrane contact sites (MCS). How MCS are regulated under the dynamic cellular environment remains poorly understood. Here, we investigate how Pex30, a membrane protein localized to the endoplasmic reticulum (ER), regulates multiple MCS in budding yeast. We show that Pex30 is critical for the integrity of ER MCS with peroxisomes and vacuoles. This requires the Dysferlin domain (DysF) on Pex30 cytosolic tail. This domain binds to phosphatidic acid (PA) both in vitro and in silico, and it is important for normal PA metabolism in vivo . The DysF domain is evolutionarily conserved and may play a general role in PA homeostasis across eukaryotes. We further show that ER-Vacuole MCS requires Pex30 C-terminal Domain of Unknown Function and that its activity is controlled by phosphorylation in response to metabolic cues. These findings provide new insights into the dynamic nature of MCS and their coordination with cellular metabolism.

cell biology↗

bioRxiv2024

TTF2 promotes replisome eviction from stalled forks in mitosis

Cells entering mitosis with incompletely replicated DNA face catastrophic chromosome segregation failure. During interphase, the replisome-associated E3 ubiquitin ligase TRAIP ubiquitylates barriers in front of the fork to allow replisome progression. In mitosis, TRAIP is reprogrammed from a trans-acting to a cis-acting ligase that can ubiquitylate the replisome itself. This enables the processing of unreplicated DNA by promoting replisome disassembly, fork breakage, and joining of the broken chromosome arms. Here, we describe a mechanism for this reprogramming: the ATPase TTF2 is recruited to the replisome, where its non-catalytic N-terminal domain tethers Cyclin B-CDK1-phosphorylated TRAIP to the leading strand DNA polymerase {varepsilon} in a geometry that allows replisome ubiquitylation. Thus, a phospho-regulated architectural switch alters replisome organization in mitosis to safeguard genome integrity before chromosome segregation.

biochemistry↗

bioRxiv2024

Aneuploidy-induced proteostasis disruption impairsmitochondrial functions and mediates aggregation ofmitochondrial precursor proteins through SQSTM1/p62

Aberrant chromosomal content, or aneuploidy, profoundly affects cellular physiology. Even a gain of a single chromosome disrupts proteostasis due to overexpression of numerous proteins. Consequently, cells accumulate SQSTM1/p62-positive cytosolic bodies and show altered proteasomal and lysosomal activity. To elucidate the p62 interaction network in aneuploid cells, we conducted p62 immunoprecipitation and proximity labeling assays followed by mass spectrometry analysis. Our investigation revealed the enrichment of mitochondrial proteins within the cytosolic p62 interactome and proxitome in aneuploid cells, but not in the proxitome spatially confined to autophagosomes. Immunofluorescence microscopy confirmed increased colocalization of p62 with novel interactors and with mitochondrial proteins in polysomic cells. Moreover, we observed mitochondrial defects characterized by increased perinuclear clustering, reduced oxygen consumption, and reduced mitochondrial DNA abundance in polysomic cells. Furthermore, we demonstrate that polysomic cells exhibit reduced import of mitochondrial proteins and accumulation of mitochondrial precursor proteins in the cytosol. Our data suggest that proteotoxic stress induced by chromosome gains leads to the sequestration of mitochondrial precursor proteins into cytosolic p62-bodies and compromises mitochondrial function.

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↗

bioRxiv2023

TopBP1 utilises a bipartite GINS binding mode to activate the replicative helicase

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Pol{varepsilon}) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Pol{varepsilon} subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.

molecular biology↗

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

Single-cell transcription profiles in Bloom syndrome patients link BLM deficiency with altered condensin complex expression signatures

Bloom syndrome (BS) is an autosomal recessive disease clinically characterized by primary microcephaly, growth deficiency, immunodeficiency, and predisposition to cancer. It is mainly caused by biallelic loss-of-function mutations in the BLM gene, which encodes the BLM helicase, acting in DNA replication and repair processes. Here, we describe the gene expression profiles of three BS fibroblast cell lines harboring causative, biallelic truncating mutations obtained by single-cell (sc) transcriptome analysis. We compared the scRNA transcription profiles from three BS patient cell lines to two age-matched wild-type controls and observed specific deregulation of gene sets related to the molecular processes characteristically affected in BS, such as mitosis, chromosome segregation, cell cycle regulation, and genomic instability. We also found specific upregulation of genes of the Fanconi anemia pathway, in particular FANCM, FANCD2, and FANCI, which encode known interaction partners of BLM. The significant deregulation of genes associated with inherited forms of primary microcephaly observed in our study might explain in part the molecular pathogenesis of microcephaly in BS, one of the main clinical characteristics in patients. Finally, our data provide first evidence of a novel link between BLM dysfunction and transcriptional changes in condensin complex I and II genes. Overall, our study provides novel insights into gene expression profiles in BS on a single-cell level, linking specific genes and pathways to BLM dysfunction.

genetics↗

bioRxiv2021

Scaling of cellular proteome with ploidy

Ploidy changes are frequent in nature and contribute to evolution, functional specialization and tumorigenesis (1,2). Analysis of model organisms of different ploidies revealed that increased ploidy leads to an increase in cell and nuclear volume, reduced proliferation (2-4), metabolic changes (5), lower fitness (6,7), and increased genomic instability (8,9), but the underlying mechanisms remain poorly understood. To investigate how the gene expression changes with cellular ploidy, we analyzed isogenic series of budding yeasts from 1N to 4N. We show that mRNA and protein abundance scales allometrically with ploidy, with tetraploid cells showing only threefold increase in proteins compared to haploids. This ploidy-specific scaling occurs via decreased rRNA and ribosomal protein abundance and reduced translation. We demonstrate that the Tor1 activity is reduced with increasing ploidy, which leads to rRNA gene repression via a novel Tor1-Sch9-Tup1 signaling pathway. mTORC1 and S6K activity are also reduced in human tetraploid cells and the concomitant increase of the Tup1 homolog Tle1 downregulates the rDNA transcription. Our results revealed a novel conserved mTORC1-S6K-Tup1/Tle1 pathway that ensures proteome remodeling in response to increased ploidy.

cell biology↗

bioRxiv2021

Systems approaches identify the consequences ofmonosomy in somatic human cells

Chromosome loss that results in monosomy is detrimental to viability, yet, it is frequently observed in cancers. How cancers survive with monosomy is unknown. Using p53 deficient monosomic cell lines, we found that chromosome loss impairs proliferation and genomic stability. Transcriptome and proteome analysis revealed a partial compensation of the gene dosage changes that mitigates the effects of chromosome loss. Monosomy triggers global gene expression changes that differ from the effects of trisomy. We show that ribosome biogenesis and translation were commonly downregulated in monosomic cells, likely due to haploinsufficiency of ribosomal genes. The ensuing ribosome biogenesis stress triggers the p53 pathway and G1 arrest when TP53 is reintroduced into monosomic cells. Accordingly, impaired ribosome biogenesis and p53 inactivation are associated with monosomy in cancer. Our first systematic study of monosomy in human cells explains why monosomy is so detrimental and how loss of p53 enables its incidence in cancer.

systems biology↗

bioRxiv2021

Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection.

Eukaryotic cells employ three SMC complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

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↗

bioRxiv2019

The proteomic inventory reveals the chloroplast ribosome as nexus within a diverse protein network

Chloroplast gene expression is tightly regulated and majorly controlled on the level of protein synthesis. Fine-tuning of translation is vital for plant development, acclimation to environmental challenges and for the assembly of major protein complexes such as the photosynthesis machinery. However, many regulatory mediators and the interaction network of chloroplast ribosomes are not known to date. We report here on a deep proteomic analysis of the plastidic ribosome interaction network in Chlamydomonas reinhardtii cells. Affinity-purification of ribosomes was achieved via endogenous affinity tagging of the chloroplast-encoded protein Rpl5, yielding a specific enrichment of >650 chloroplast-localized proteins. The ribosome interaction network was validated for several proteins and provides a new source of mainly conserved factors directly linking translation with central processes such as protein folding, photosystem biogenesis, redox control, RNA maturation, energy and metabolite homeostasis. Our approach provided the first evidence for the existence of a plastidic co-translational acting N-acetyltransferase (cpNAT1). Expression of tagged cpNAT1 confirmed its ribosome-association, and we demonstrated the ability of cpNAT1 to acetylate substrate proteins at their N-terminus. Our dataset establishes that the chloroplast protein synthesis machinery acts as nexus in a highly choreographed, spatially interconnected protein network and underscores its wide-ranging regulatory potential during gene expression. ONE-SENTENCE SUMMARYAffinity purification of Chlamydomonas reinhardtii chloroplast ribosomes and subsequent proteomic analysis revealed a broad spectrum of interactors ranging from global translation control to specific pathways.

biochemistry↗