bims-mecosi 2026-05-10 papers

bims-mecosi

Biomed News

on Membrane contact sites

Issue of 2026–05–10
twelve papers selected by
Verena Kohler, Umeå University

Gypenoside XLIX and Mitochondria-Associated ER Membranes in Non-Alcoholic Fatty Liver Disease: Mechanistic Insights and Emerging Perspectives.
RYR:ATP6V0A1 complexes couple ER-lysosome contact sites to dynamic autophagy control.
A MAM-targeting therapeutic peptide restores autophagy homeostasis and ameliorates atherosclerosis.
Proteasome-dependent degradation and nucleus-vacuole junctions sustain proteostasis during acute glucose starvation.
Organelle-orchestrated cGAS-STING signaling and its role in neurodegeneration.
Unraveling new functions of the Ca2+ sensor STIM1 in cell signaling.
Global mitochondrial connectivity map reveals the landscape of yeast functional assemblies and conserved protein communities.
Pridopidine Protects ALS Patient-Derived Neural Progenitor Cells via Sigma-1 Receptor Activation.
Mitochondrially tethered Mmm1 can function as a sole lipid transporter at ER-mitochondria contacts.
PACS2 Alleviates Sepsis-Induced Myopathy by Activating ERK-MAPK Signalling Pathway to Suppress ER-Phagy.
Dynamic proximity networks of myosin-19 (Myo19) and its mitochondrial receptors Miro2 and metaxin-3.
Rewiring Mitochondrial Phosphatidylethanolamine Metabolism Identifies New and Unaccounted Trafficking Steps.

Molecules. 2026 Apr 17. pii: 1325. [Epub ahead of print]31(8):

Gypenoside XLIX and Mitochondria-Associated ER Membranes in Non-Alcoholic Fatty Liver Disease: Mechanistic Insights and Emerging Perspectives.

Xinyi Kwan, Muhammad Shahzad Aslam, Huiqing Liang, Shaodong Chen.

  Gypenoside XLIX is a bioactive saponin with reported diverse biological activities, including antioxidant, regulation of cell growth, immune responses, and metabolic regulatory properties. The increasing global prevalence of non-alcoholic fatty liver disease (NAFLD) underscores the importance of exploring novel therapeutic agents such as Gypenoside XLIX. NAFLD pathogenesis involves lipotoxicity, oxidative stress, and mitochondrial dysfunction, in which mitochondria-associated endoplasmic reticulum membranes (MAMs) play a critical role in organelle communication, calcium signaling, and lipid metabolism. This narrative review summarizes current evidence indicating that Gypenoside XLIX may modulate oxidative stress, restore mitochondrial membrane potential, and regulate calcium homeostasis, thereby indirectly influencing MAM integrity and function. These effects can reduce lipid accumulation, improve hepatocellular metabolism, and attenuate inflammatory responses. This review evaluates the mechanistic impact and function of Gypenoside XLIX on MAM integrity and its effects on NAFLD. However, there is limited direct experimental evidence linking Gypenoside XLIX to MAM regulation, and further studies are required to validate its mechanisms and therapeutic potential in clinical settings.

Keywords:  Gypenoside XLIX; calcium homeostasis; mitochondria-associated endoplasmic reticulum membranes; mitochondrial dysfunction; non-alcoholic fatty liver disease

DOI:  https://doi.org/10.3390/molecules31081325
Autophagy. 2026 May 05.

RYR:ATP6V0A1 complexes couple ER-lysosome contact sites to dynamic autophagy control.

Jens Loncke, Manon Callens, Geert Bultynck, Tim Vervliet.

  Ryanodine receptors (RYRs) are ER-resident Ca2 + -release channels enriched in excitable cells, including neurons. RYR hyperactivity is implicated in early pathogenesis of disorders such as Alzheimer's disease (AD), which is associated with impaired autophagy. We recently uncovered a mechanism linking RYR activity to lysosome availability for autophagy. RYRs localize to ER - lysosome contact sites via direct binding to ATP6V0A1, a V-ATPase subunit that also suppresses RYR-mediated Ca2 + release. In human iPSC-derived cortical neurons, spontaneous RYR activity promotes lysosomal secretion, depleting the intracellular lysosomal pool and inhibiting autophagic flux. RYR inhibition promotes ER - lysosome contacts, limits lysosomal secretion, and restores lysosome availability for autophagosome fusion and cargo degradation (including APP). Conversely, disrupting the RYR:ATP6V0A1 interaction using a RYR-derived protein fragment serving as a "decoy" for ATP6V0A1 evokes RYR hyperactivity and stimulates lysosomal secretion. In this Punctum, we discuss how this RYR2:ATP6V0A1 "contact-site hub" may be perturbed in disease and highlight open questions on how lysosomes decode RYR-derived Ca2 + signals.

Keywords:  Calcium signaling; V-type ATPase; endoplasmic reticulum; lysosome; membrane contact site; ryanodine receptor

DOI:  https://doi.org/10.1080/15548627.2026.2669981
Theranostics. 2026 ;16(11): 6113-6131

A MAM-targeting therapeutic peptide restores autophagy homeostasis and ameliorates atherosclerosis.

Jungmin Ha, Minjeong Ko, Yong-Beom Lim, Ho Jeong Kwon.

   Rationale: Mitochondria-associated ER membranes (MAMs) are critical hubs for Ca2+ signaling, energy homeostasis, and autophagy. Their dysregulation contributes to lipid-driven cardiovascular diseases; however, selective and reversible strategies to modulate MAM-associated protein-protein interactions (PPIs) remain limited. This study aimed to develop a targeted peptide to disrupt the IP3R-GRP75-VDAC1 complex and evaluate its therapeutic efficacy in atherosclerosis.
Methods: Based on structural and interface analyses of the IP3R-GRP75 complex, we designed cell-permeable MAM-targeting peptides. The activity of the lead candidate, Peptide 4, was assessed using proximity ligation assays, microscale thermophoresis (MST) analysis, cellular thermal shift assays, co-immunoprecipitation, live-cell Ca2+ imaging, and autophagy flux analyses in endothelial cells and macrophages under basal and oxidized low-density lipoprotein (oxLDL)-induced stress. The therapeutic efficacy was further evaluated in Western diet-fed ApoE-/- mice.
Results: Peptide 4 bound to GRP75, disrupted the IP3R-GRP75 interaction, and selectively attenuated ER-to-mitochondria Ca2+ transfer. This controlled Ca2+ modulation modestly reduced cellular ATP levels, activated the AMPK-TFEB axis, and restored functional autophagic flux. These effects were preserved under oxLDL-induced lipid stress. Restoration of MAM architecture closely correlated with autophagy recovery and lipid clearance, indicating its potential utility as a pharmacodynamic indicator. In vivo, systemic administration of Peptide 4 significantly improved serum lipid profiles, attenuated aortic plaque formation, reduced cardiac lipid deposition, and normalized MAM architecture in ApoE-/- mice.
Conclusions: Our findings identify peptide-mediated targeting of the IP3R-GRP75 interaction as a promising strategy to modulate MAM structure, activate adaptive autophagy, and alleviate atherosclerotic pathology. This study supports organelle contact site modulation as both a therapeutic mechanism and a measurable disease-responsive feature, highlighting peptide-based modulation of protein-protein interactions as a promising approach for metabolic and cardiovascular diseases.

Keywords:  Atherosclerosis; Mitochondria-associated ER membranes; Organelle crosstalk; Peptide therapeutics; Protein-protein interaction

DOI:  https://doi.org/10.7150/thno.132357
bioRxiv. 2026 Apr 25. pii: 2026.04.22.720209. [Epub ahead of print]

Proteasome-dependent degradation and nucleus-vacuole junctions sustain proteostasis during acute glucose starvation.

Mihaela Pravica, Dina Franić, Matko Bazdan, Dominik Guzalić, Antonio Bedalov, Mirta Boban.

How protein quality control is maintained during acute metabolic stress remains poorly understood. In budding yeast, abrupt glucose depletion rapidly lowers ATP levels and leads to the formation of chaperone-containing inclusions, suggesting that ATP-dependent degradation of misfolded proteins may be compromised when energy becomes limiting. Here we find that selective degradation of misfolded proteins remains active during acute glucose starvation despite reduced cellular ATP levels. Using model misfolded substrates in yeast Saccharomyces cerevisiae , we show that misfolded proteins continue to be efficiently degraded throughout both early and late phases of acute glucose depletion. This degradation requires the proteasome and depends on its functional 19S regulatory particle, indicating that ATP-dependent proteasomal activity persists during metabolic stress. We further find that nucleus-vacuole junctions (NVJs) promote efficient degradation during prolonged glucose starvation, revealing a role for organelle contact sites in supporting proteostasis under energy limitation. Together, these findings indicate that cells preserve proteasome-mediated proteostasis during acute glucose starvation, while NVJ membrane contact sites help sustain degradation capacity when metabolic resources are scarce.

DOI: https://doi.org/10.64898/2026.04.22.720209
Pharmacol Res. 2026 May 01. pii: S1043-6618(26)00131-3. [Epub ahead of print] 108216

Organelle-orchestrated cGAS-STING signaling and its role in neurodegeneration.

Jin-Yue Liu, Sheng-Ye Liu, Li-Xian Ran, Wen-Hao Qiao, Fan Zhang, Kai-Song Zhang, Xu-Xin Liang, Ming-Yan Liu, Zhi-Hua Yu, Min-Jie Wei, Xin Zhong.

  The cGAS-STING signaling pathway serves as a central signalling axis of the innate immune system, and its aberrant activation plays a pivotal role in inflammatory responses. Recent studies have demonstrated that its regulation depends not only on individual organelles but also on a coordinated interorganelle network. This review systematically analyze how mitochondria, centrosomes, the endoplasmic reticulum (ER), membrane contact sites (MCSs), the Golgi apparatus, endosomes, and lysosomes collectively orchestrate cGAS-STING signaling. Mitochondria initiate signaling by releasing mitochondrial DNA; centrosomes serve as platforms for double-stranded DNA accumulation to potentiate cGAS activation; the ER anchors STING in a calcium homeostasis-dependent manner; mitochondrial-associated ER membranes (MAMs) integrate calcium and lipid signaling as regulatory checkpoints governing STING trafficking to the Golgi apparatus; the Golgi amplifies downstream signaling through site-specific post-translational modifications of STING; finally, the endosome-lysosome system, together with ER-lysosome MCSs, acts as a coordinated hub for STING sorting, lysosomal degradation and signal termination. Consequently, disruption of organelle homeostasis leads to persistent STING activation. In neurodegenerative conditions including Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis and Huntington's disease, organelle dysfunction resulting from calcium overload, impaired organelle clearance, proteolytic cleavage of tethering proteins or multi-source attacks drives aberrant STING signaling. Sustained STING activity exacerbates pathological cascades such as protein misfolding, chronic neuroinflammation, and progressive neuronal loss. Therefore, therapeutic strategies targeting key regulatory nodes of the STING pathway, from upstream organelle repair to direct pharmacological inhibition, offer significant potential to mitigate disease-associated pathological progression and constitute a promising foundation for precision therapeutics in neurodegenerative disorders.

Keywords:  Neurodegenerative diseases; Organelle interactions; Organelle regulation; Therapeutic strategies; cGAS-STING signaling pathway

DOI:  https://doi.org/10.1016/j.phrs.2026.108216
Biochem Soc Trans. 2026 05 27. 54(5): 437-447

Unraveling new functions of the Ca2+ sensor STIM1 in cell signaling.

Irene Sanchez-Lopez, Yolanda Orantos-Aguilera, Aida M Lopez-Guerrero, Eulalia Pozo-Guisado, Francisco Javier Martin-Romero.

  Calcium (Ca2+) signaling is a fundamental regulator of virtually all aspects of eukaryotic cell physiology, including gene expression, secretion, metabolism, motility, and cell fate decisions. The spatial and temporal control of cytosolic Ca2+ signals relies on a coordinated interplay between intracellular Ca2+ stores and plasma membrane (PM) Ca2+ channels. A critical advance in this field over the past two decades was the molecular identification of stromal interaction molecule 1 (STIM1) as the long-sought Ca2+ sensor that couples depletion of endoplasmic reticulum Ca2+ stores to Ca2+ influx across the PM. STIM1 has been established as a core component of store-operated Ca2+ entry, acting through direct activation of ORAI Ca2+ channels. However, accumulating evidence now indicates that STIM1 functions extend beyond this canonical role. STIM1 participates in the regulation of multiple classes of ion channels, contributes to the organization of membrane contact sites, and acts as a signaling scaffold influencing cellular processes independently of classical store depletion. This review summarizes the discovery and canonical functions of STIM1 and focuses on its emerging non-canonical roles, highlighting how STIM1 has evolved from an ER Ca2+ sensor into a multifunctional signaling hub.

Keywords:  DNA damage response; calcium signalling; endoplasmic reticulum; immune response; mitochondria; mitochondrial dysfunction

DOI:  https://doi.org/10.1042/BST20250458
Nat Commun. 2026 May 05.

Global mitochondrial connectivity map reveals the landscape of yeast functional assemblies and conserved protein communities.

Matthew Jessulat, Sadhna Phanse, Hiroyuki Aoki, Kirsten Broderick, Qingzhou Zhang, Inês Gomes Castro, Noelle Alexa Novales, Sakib Abrar Hossain, Tatiana Saccon, Mohamed Taha Moutaoufik, Thomson Patrick Joseph, Shahreen Amin, Larrisa Hoell, Zoran Minic, Yury Bykov, Noga Preminger, Sahily Gonzalez Crespo, Jamie Snider, Ashkan Golshani, Igor Stagljar, Jose R Rodriguez-Medina, Catherine F Clarke, Maya Schuldiner, Mohan Babu.

Mitochondria are essential organelles whose functions depend on coordinated multiprotein complexes, yet their composition and organization remain incomplete. Here, we present a large-scale map of mitochondrial protein complexes by integrating affinity purification of 740 endogenously GFP-tagged mitochondrial proteins with biochemical co-fractionation of mitochondrial extracts from yeast (Saccharomyces cerevisiae) grown under respiratory conditions. Mass spectrometry identifies 13,716 high-confidence protein associations and defines 556 heteromeric complexes, many previously unknown. These assemblies reveal factors involved in coenzyme Q6 biosynthesis, membrane contact sites, phospholipid transport, and coordination with the MICOS complex during respiration. We further link 538 assemblies to 294 candidate human disease genes and construct a conservation map of 852,146 predicted mitochondrial interactions across 271 genomes, and validate key predictions in human cell lines and mouse brain tissue. Together, this work provides a comprehensive mitochondrial interactome, assigning functions to poorly characterized proteins, and offering insights into mitochondrial biology and disease-associated assemblies.

DOI: https://doi.org/10.1038/s41467-026-72525-2
Int J Mol Sci. 2026 Apr 14. pii: 3489. [Epub ahead of print]27(8):

Pridopidine Protects ALS Patient-Derived Neural Progenitor Cells via Sigma-1 Receptor Activation.

May Meltzer, Maya Shefler Zamir, Noam Tzuri, Andrew M Tan, Michal Geva, Michael R Hayden, Rachel G Lichtenstein.

  The sigma-1 receptor (S1R) is an endoplasmic reticulum (ER)-resident protein enriched at the mitochondria-associated ER membranes (MAMs) that supports ER homeostasis, preserves mitochondrial function, and enhances cell survival under stress. Disruptions of MAM integrity and prolonged ER stress are well-recognized pathological features of amyotrophic lateral sclerosis (ALS), contributing to motor neuron dysfunction and degeneration. In this study, we evaluated the protective effects of pridopidine, a highly selective and potent S1R agonist currently in clinical development for Huntington's disease (HD) and ALS, using neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs) from a patient with sporadic ALS. Exposure of ALS NPCs to the ER stressor tunicamycin increased the ER stress markers binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), disrupted mitochondrial membrane potential, upregulated expression of the mitochondrial apoptotic marker, BAX, increased caspase-3 activation, and reduced cell viability. Pridopidine significantly attenuated tunicamycin-induced BiP and CHOP expression in a biphasic, dose-dependent manner (with maximal efficacy at 1 µM), consistent with the typical pharmacology of S1R agonists. Pridopidine restored mitochondrial membrane potential, reduced mitochondrial apoptotic signaling, shown by decreased BAX expression and caspase-3 activation, and improved survival of ALS-NPCs under ER stress. Co-treatment with the selective S1R antagonist, NE-100, attenuated these effects, supporting an S1R-mediated mechanism of action for pridopidine. Together, these results demonstrate that S1R activation by pridopidine mitigates ER-stress-induced mitochondrial dysfunction and cell loss in ALS-NPCs, resulting in enhanced survival of NPCs supporting the therapeutic potential of pridopidine in ALS.

Keywords:  Sigma-1 receptor; amyotrophic lateral sclerosis; endoplasmic reticulum stress; iPSC-derived neural progenitor cells; mitochondria-associated membranes; mitochondrial membrane potential; pridopidine

DOI:  https://doi.org/10.3390/ijms27083489
J Cell Biol. 2026 Jul 06. pii: e202411196. [Epub ahead of print]225(7):

Mitochondrially tethered Mmm1 can function as a sole lipid transporter at ER-mitochondria contacts.

Christian Covill-Cooke, Takashi Hirashima, Shin Kawano, Joe Ganellin, Andrew Moody, Sabine N S van Schie, Arun T John Peter, Chika Horie Saito, Toshiya Endo, Benoît Kornmann.

Yeast mitochondria receive the majority of their lipids from the ER via the heterotetrameric ERMES lipid transport complex. This complex is thought to establish a lipid-transporting bridge of fixed composition spanning the space between both organelles. Intriguingly, however, some of the lipid-transporting components of the complex can be replaced by an artificial ER-mitochondria tether without lipid transport activity, questioning ERMES' relevance in lipid transport. Here, we show that Mmm1, one of the four ERMES subunits, alone is sufficient to support ERMES function when it is artificially tethered to mitochondria, provided its lipid-binding domain is intact. Combined with our previous finding that the absence of Mdm12 and Mdm34 can be rescued by the presence of Mmm1 and the artificial tethering protein ChiMERA, our results suggest that Mmm1 can act as the sole lipid transporter at the ER-mitochondrial contact sites, provided that Mdm10 is present, even in the absence of the other two subunits. Thus, our work reconciles ERMES' importance in lipid transport with the fact that the lipid transport activity of some of its components is not strictly necessary for function.

DOI: https://doi.org/10.1083/jcb.202411196
J Cachexia Sarcopenia Muscle. 2026 Jun;17(3): e70308

PACS2 Alleviates Sepsis-Induced Myopathy by Activating ERK-MAPK Signalling Pathway to Suppress ER-Phagy.

Xuexin Li, Zu-An Shi, Fei He, Guo Mu, Feixiang Wang, Bowen Sun, Xiaobin Wang, Li Liu.

   BACKGROUND: Sepsis-induced myopathy (SIM) is a common and life-threatening complication, but its underlying mechanisms remain poorly understood. PACS2, a key resident protein at mitochondria-associated endoplasmic reticulum membranes (MAMs), regulates ER homeostasis under various pathological conditions. However, whether sepsis disrupts PACS2-dependent MAM integrity, thereby triggering ER dysfunction and muscle wasting, remains unexplored.
METHODS: We established a sepsis mouse model via cecal ligation and puncture (CLP) and assessed muscle function using compound muscle action potential (CMAP) recording and grip strength measurements. Muscle atrophy was evaluated by H&E staining and Western blotting. PACS2 expression was determined by Western blotting, immunohistochemistry and qRT-PCR. MAM integrity was assessed by immunofluorescence co-localization of IP3R and VDAC1, and ER-phagy (reticulophagy) activation was evaluated by transmission electron microscopy, Western blotting and fluorescence microscopy. To investigate the functional role of PACS2, adeno-associated virus (AAV)-mediated PACS2 overexpression was performed in mouse tibialis anterior muscle and gastrocnemius muscles, followed by RNA-sequencing analysis. The MAPK pathway proteins p-ERK, p-P38 and p-JNK levels were assessed by Western blotting, and the involvement of ERK-MAPK signalling was tested pharmacologically via intraperitoneal injection of the ERK inhibitor SCH772984.
RESULTS: Septic mice developed progressive skeletal muscle atrophy (p < 0.001) and dysfunction (p < 0.01), accompanied by 56% reduction in PACS2 expression at 96 h post-CLP (p < 0.01), 25% decrease in MAM integrity (p < 0.05) and subsequent activation of FAM134B-mediated ER-phagy (p < 0.01). AAV-mediated PACS2 overexpression significantly alleviated muscle atrophy by restoring MAM integrity by 28% (p < 0.01), reducing FAM134B expression by 43% (p < 0.01) and attenuating ER-phagy (p < 0.01). Co-immunoprecipitation revealed no detectable direct protein-protein interaction between PACS2 and FAM134B. Transcriptome sequencing and Western blotting analysis demonstrated that PACS2 overexpression specifically activated the ERK-MAPK signalling pathway (55% increase in p-ERK, p < 0.01) without affecting p-P38 or p-JNK levels (p>0.05), which suppressed FAM134B-mediated ER-phagy (p < 0.05) and ameliorated muscle atrophy (p < 0.05) by inhibiting nuclear translocation of TFEB (p < 0.01). Pharmacological ERK inhibition with SCH772984 abolished the protective effects of PACS2 by promoting TFEB nuclear translocation (p < 0.001) and TFEB-mediated FAM134B expression (p < 0.001).
CONCLUSIONS: Our findings demonstrate that SIM is closely associated with disrupted MAM integrity. PACS2 plays a critical role in maintaining MAM structural integrity and regulating FAM134B-mediated ER-phagy through the ERK-MAPK-TFEB signalling axis, thereby providing novel mechanistic insights and potential therapeutic targets for SIM.

Keywords:  ERK–MAPK signalling pathway; FAM134B; MAM; PACS2; sepsis‐induced myopathy

DOI:  https://doi.org/10.1002/jcsm.70308
Mol Cell Proteomics. 2026 May 06. pii: S1535-9476(26)00078-2. [Epub ahead of print] 101582

Dynamic proximity networks of myosin-19 (Myo19) and its mitochondrial receptors Miro2 and metaxin-3.

Aya Attia, Jürgen Eirich, Ulrike Honnert, Petra Nikolaus, Birgit Lohmann, Iris Finkemeier, Martin Bähler.

  Myosin-19 (Myo19) plays a crucial role in mitochondrial dynamics, cristae organization and ER-mitochondria contact sites (ERMCS). It regulates cytokinesis and inheritance of mitochondria to daughter cells. To better understand the dynamic molecular network of Myo19 during the cell cycle, we determined the in vivo proximity protein interaction networks of Myo19 in interphase and prometaphase using proximity-based TurboID biotinylation followed by mass spectrometry. We further determined the proximity networks of its known mitochondrial binding partners Miro2 and metaxin-3. The outer mitochondrial membrane protein Miro2 not only binds but also stabilizes Myo19. This interaction depends on the nucleotide state of the N-terminal GTPase domain of Miro2. Therefore, we analysed both the proximity networks of Miro2 and its GTP-binding mutant Miro2 T18N. We were able to show a differential association of Myo19 during the cell cycle with functional protein clusters and a participation of Myo19 in mitochondrial trafficking, ERMCS and mitochondria intermembrane space bridging complex / cristae organizing system (MIB/MICOS). The proximity network of Myo19 showed more overlap with Miro2 than metaxin-3. Abolishing GTP-binding to the N-terminal GTPase domain of Miro2 reduced the number of proteins in proximity of Miro2 considerably. In conclusion, we discovered a comprehensive dynamic in vivo protein proximity network of Myo19 and its mitochondrial receptors Miro2 and metaxin-3.

Keywords:  ERMCS; MIB/MICOS; MTX3; Miro2; Mitochondrial dynamics; Myo19; OMM; TurboID Proximity Labelling; actin; mitochondria; myosin

DOI:  https://doi.org/10.1016/j.mcpro.2026.101582
bioRxiv. 2026 Apr 24. pii: 2026.04.22.720193. [Epub ahead of print]

Rewiring Mitochondrial Phosphatidylethanolamine Metabolism Identifies New and Unaccounted Trafficking Steps.

Rashima Prem, Erica Avery, Juliana M Marquez, Chi Xie, Steven M Claypool.

The distinct compositions of the two mitochondrial membranes are generated through a combination of phospholipids that mitochondria can make and those they take; both processes depend on a series of distinct lipid trafficking steps. Mitochondria make phosphatidylethanolamine (PE) through the action of the phosphatidylserine decarboxylase Psd1, an intermembrane space (IMS)-facing integral inner membrane (IM) protein. Psd1 has been proposed to act on its endoplasmic reticulum-derived substrate, phosphatidylserine (PS), after its transport to the mitochondrial outer membrane (OM) and either following its Ups2/Mdm35-mediated transport across the IMS to the IM or instead, on the IMS-side of the OM in a process enabled by the mitochondrial contact site and cristae organizing system (MICOS). Here, we implement a two-pronged Psd1 rewiring-based strategy predicted to either 1) circumvent the need for Ups2/Mdm35 and/or MICOS; or 2) selectively ablate the ability of Psd1 to work in trans . Our results with yeast harboring Psd1 targeted to the OM demonstrate that, with respect to mitochondrial PE production, Ups2/Mdm35 and MICOS indeed function within the IMS. Using yeast expressing a topologically inverted Psd1 chimera that faces the matrix, we identify previously unappreciated transbilayer lipid trafficking steps within the IM and show that Psd1 does not operate via a MICOS-organized in trans mechanism. Further, retained flux through inverted Psd1 when both Ups2/Mdm35 and MICOS are absent strongly implicates the existence of a major, yet presently unknown, mediator(s) of lipid movement across the IMS. Collectively, these data suggest a new model of how mitochondrial membrane diversity is established and maintained.

DOI: https://doi.org/10.64898/2026.04.22.720193