Biology subjects

von der Malsburg, K.

Publications and source records attributed to von der Malsburg, K..

9 recordsLinked to original sources

bioRxiv2026

Exploring the activation mechanism of the Mic60 subcomplex governing mitochondrial crista junctions

Crista junctions (CJs) are narrow infoldings of the inner mitochondrial membrane, serving as entry points into the cristae microenvironment. The mitochondrial contact site and cristae organizing system (MICOS) complex is a highly conserved protein machinery controlling CJ biogenesis and architecture, but its assembly mechanism driving CJ formation has remained unclear. By determining a crystal structure of a Chaetomium thermophilum Mic60-Mic19 complex and combining it with modelling and mutagenesis studies, we identify an extended helical region (EHR) of the Mic60 mitofilin domain and a further Mic19 helix as crucial elements of complex formation. In vitro cross-linking data and hydrogen-deuterium exchange mass spectrometry resolve domain assembly and conformational dynamics of the Mic60 Mic19 subcomplex and point to the EHR as a molecular switch controlling Mic60 transition from a closed dimer to a Mic19-bound hetero-octamer. Our data provide a structural model for Mic60 activation and therefore MICOS assembly as a prerequisite for crista junction formation.

molecular biology↗

bioRxiv2026

Super-resolution complexome of human mitochondria elucidates translocase, morphology and OXPHOS networks

Mitochondria function as cellular powerhouses and central hubs in metabolism, redox and stress reactions, signaling and apoptosis1-5. Defects of mitochondria lead to numerous human diseases1,6-8. The integration of mitochondrial proteins into complexes and networks is crucial for their function. Whereas the composition of the human mitochondrial proteome has been studied8,9, only limited information is available on the organization of the proteome into protein complexes and assemblies. Here we present a systematic mapping of the human mitochondrial complexome from HEK293T cells at super-resolution, resolving more than 7,000 abundance profile peaks of mitochondrial proteins. Proteins functioning in signaling, cell stress, protein biogenesis, turnover and membrane dynamics display particularly high complexities. High resolution and precise quantification enable discrimination between canonical constituents and non-stoichiometric regulatory interactors of the ATP synthase, major metabolite channels and import translocases. The complexome reveals membrane-spanning networks of protein insertase and morphology machinery, and co-assembly of protein import and export components at the major respiratory supercomplex, unraveling a multifunctional organization of mitochondrial machineries. This complexome represents a fully interactive resource for the systematic analysis of human mitochondrial machineries and interaction networks.

biochemistry↗

bioRxiv2025

Structural studies suggest CCDC127 as a novel membrane contact site protein in the mitochondrial intermembrane space

Mitochondria feature a sophisticated membrane architecture, with a planar mitochondrial outer membrane (MOM) and a folded inner membrane (MIM). Due to the remarkable adaptability of mitochondria, a proteinaceous network in the intermembrane space (IMS) was proposed to confer both stability and flexibility. However, components of such scaffolds, tentatively termed the mitoskeleton, have remained largely elusive. The mitochondrial contact site and organizing system (MICOS), a central organizer of mitochondrial membrane architecture, was suggested to participate in mitoskeleton formation. Here, we structurally characterize the coiled-coil domain-containing 127 (CCDC127) protein, a putative interactor of MICOS. We show that CCDC127s amino-terminal transmembrane region is anchored in the MOM and the bulk soluble part exposed to the IMS. A crystal structure of CCDC127s central coiled-coil displays a parallel dimer which further oligomerizes into tetramers. We demonstrate that the carboxy-terminal helical bundle (CHB) domain dimerizes to create a peripheral membrane-binding site. Supported by electron microscopy data, we propose a structural model of CCDC127 as intramitochondrial membrane contact site protein mediating the structural organization of the IMS as part of the mitoskeleton.

molecular biology↗

bioRxiv2025

Topology control by a conserved cysteine pair in the OMM-protein CCDC127 enables MICOS interaction

Mitochondrial disulfide relay substrates beyond the canonical substrates remain incompletely defined. Revisiting the human MIA40 interactome with enhanced depth, we identified CCDC127 as a previously unrecognized substrate candidate. CCDC127 contains a single transmembrane segment and a conserved C-terminal helical bundle domain (CHB). Comprehensive proteomic and biochemical analyses revealed that, contrary to earlier reports, CCDC127 adopts an Nout-Cin topology in the outer mitochondrial membrane (OMM) with its CHB residing in the intermembrane space (IMS). CCDC127 undergoes oxidation by the disulfide relay, forming a long-range intramolecular disulfide bond between C174 and C219. Loss of these cysteines disrupts correct OMM insertion, inverts transmembrane topology and triggers proteasome-dependent degradation, establishing the disulfide as a key determinant of CCDC127 maturation. Interactome analyses identified MICOS components--particularly the MIC60/MIC19 module--as major partner proteins required for the stability of large oligomeric CCDC127 complexes. CCDC127 deficiency impaired cellular proliferation, influenced phospholipid levels, and caused grossly altered cristae morphology. Together, CCDC127 emerges as a MICOS-associated OMM protein essential for mitochondrial membrane organization and lipid homeostasis.

biochemistry↗

bioRxiv2025

A multiplexed approach for genetic screening of human cells by electron microscopy uncovers a critical effector of mitochondrial cristae shape

Mitochondria form complex and diverse membrane-architectures essential for their multiple functions. Whereas critical proteins sculpting mitochondrial membranes have been identified, the molecular basis for many key features remains enigmatic. Exploration of membrane ultrastructure, in general, is limited by the tradeoff between resolution and throughput: electron microscopy (EM) is essential to resolve their ultrastructure but lacks scalability for systematic functional discovery. To overcome this limitation, we developed a high-resolution screening pipeline for EM of multiplexed human cell pools, hMultiCLEM (human Multiplexed Correlative Light and EM). To showcase the power of hMultiCLEM we performed a genetic screen exploring mitochondrial ultrastructure. hMultiCLEM confirmed proposed cristae modulators and uncovered additional ones illuminating the protein networks driving cristae organization. Validation of candidates highlighted an intermembrane space (IMS) protein linked to Menieres disease, which we named MISHA (Mitochondrial-IMS membrane-SHApe-impacting protein). More broadly, hMultiCLEM transforms the EM field, enabling genetic/chemical screening in basic and medical research.

cell biology↗

bioRxiv2025

iAPEX: Improved APEX-based proximity labeling for subcellular proteomics using an enzymatic reaction cascade

Ascorbate peroxidase (APEX) is a versatile labeling enzyme used for live-cell proteomics at high spatial and temporal resolution. However, toxicity of its substrate hydrogen peroxide and background labeling by endogenous peroxidases limit its use to in vitro studies of specific cell types. By combining APEX2 with a D-amino acid oxidase to locally produce hydrogen peroxide, we establish a more versatile, improved APEX (iAPEX) workflow that minimizes hydrogen peroxide toxicity and reduces non-specific background labeling. We employ iAPEX to perform live-cell proteomics of a cellular microdomain, the primary cilium, in previously inaccessible cell lines, leading to the identification of new ciliary proteins. Our study robustly validates common ciliary proteins across two distinct cell lines, while observed differences may reflect heterogeneity in primary cilia proteomes. iAPEX proximity labeling in Xenopus laevis provides a proof-of-concept for future in vivo applications.

biochemistry↗

bioRxiv2024

Pancreatic islets undergo functional and morphological adaptation during development of Barth Syndrome

Barth syndrome is a multisystem genetic disorder caused by mutation in TAFAZZIN, a gene that encodes a phospholipid:lysophospholipid transacylase important for cardiolipin remodeling. Barth Syndrome patients suffer from a number of symptoms including early heart failure, fatigue, and systemic metabolic alterations, including hypoglycemia. The endocrine pancreas is central to glucose homeostasis, however, the impact of defective cardiolipin remodeling on pancreatic islet function and the consequences for systemic metabolism is unclear. Surprisingly, in a mouse model with global TAFAZZIN knockdown, we observed improved glucose tolerance compared to wildtype littermates. We show that pancreatic islet metabolism and secretory function are robustly maintained through various compensatory mechanisms including increased glucose uptake and increased mitochondrial volume. Transcriptomics analyses revealed increased expression of genes encoding proteins involved in N-acetylglucosamine synthesis and protein O-linked N-acetylglucosaminylation. These pathways might provide a molecular mechanism for coupling metabolic changes to mitochondrial volume regulation.

physiology↗

bioRxiv2024

Coordination of cytochrome bc1 complex assembly at MICOS

The boundary and cristae domains of the mitochondrial inner membrane are connected by crista junctions. Most cristae membrane proteins are nuclear-encoded and inserted by the mitochondrial protein import machinery into the inner boundary membrane. Thus, they must overcome the diffusion barrier imposed by crista junctions to reach their final location. Here, we show that respiratory chain complexes and assembly intermediates are physically connected to the mitochondrial contact site and cristae organizing system (MICOS) that is essential for formation and stability of crista junctions. We identify the inner membrane protein Mar26 (Fmp10) as determinant in the biogenesis of the cytochrome bc1 complex (complex III). Mar26 couples a Rieske Fe/S protein-containing assembly intermediate to MICOS. Our data indicate that Mar26 maintains an assembly-competent Rip1 pool at crista junctions where complex III maturation likely occurs. MICOS facilitates efficient Rip1 assembly by recruitment of complex III assembly intermediates to crista junctions. We propose that MICOS, via interaction with assembly factors such as Mar26, directly contributes to the spatial and temporal coordination of respiratory chain biogenesis.

biochemistry↗

bioRxiv2022

Structural insights into crista junction formation by the Mic60-Mic19 complex

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil domain of the mitochondrial contact and cristae organizing system (MICOS) subunit Mic60 forms an elongated, bow tie-shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with curved CJ necks. Our study suggests that the Mic60-Mic19 subcomplex transverses CJs as a molecular strut, thereby controlling CJ architecture and function.

cell biology↗