Membrane binding and lipid-protein interaction of the C2 domain from coagulation factor V.

Anchoring of coagulation factors to anionic regions of the membrane involves the C2 domain as a key player. The rate of enzymatic reactions of the coagulation factors is increased by several orders of magnitude upon membrane binding. However, the precise mechanisms behind the rate acceleration remain unclear, primarily because of a lack of understanding of the conformational dynamics of the C2-containing factors and corresponding complexes. We elucidate the membrane-bound form of the C2 domain from human coagulation factor V (FV-C2) by characterizing its membrane binding the specific lipid-protein interactions. Employing all-atom molecular dynamics simulations and leveraging the highly mobile membrane-mimetic (HMMM) model, we observed spontaneous binding of FV-C2 to a phosphatidylserine (PS)-containing membrane within 2-25 ns across twelve independent simulations. FV-C2 interacted with the membrane through three loops (spikes 1-3), achieving a converged, stable orientation. Multiple HMMM trajectories of the spontaneous membrane binding provided extensive sampling and ample data to examine the membrane-induced effects on the conformational dynamics of C2 as well as specific lipid-protein interactions. Despite existing crystal structures representing presumed "open" and "closed" states of FV-C2, our results revealed a continuous distribution of structures between these states, with the most populated structures differing from both "open" and "closed" states observed in crystal environments. Lastly, we characterized a putative PS-specific binding site formed by K23, Q48, and S78 located in the groove enclosed by spikes 1-3 (PS-specificity pocket), suggesting a different orientation of a bound headgroup moiety compared to previous proposals based upon analysis of static crystal structures.

Membrane-anchoring selenophene viologens for antibacterial photodynamic therapy against periodontitis via restoring subgingival flora and alleviating inflammation.

Antibacterial photodynamic therapy (aPDT) has emerged as a promising strategy for treating periodontitis. However, the weak binding of most photosensitizers to bacteria and the hypoxic environment of periodontal pockets severely hamper the therapeutic efficacy. Herein, two novel oxygen-independent photosensitizers are developed by introducing selenophene into viologens and modifying with hexane chains (HASeV) or quaternary ammonium chains (QASeV), which improve the adsorption to bacteria through anchoring to the negatively charged cell membrane. Notably, QASeV binds only to the bacterial surface of Porphyromonas gingivalis and Fusobacterium nucleatum due to electrostatic binding, but HASeV can insert into their membrane by strong hydrophobic interactions. Therefore, HASeV exhibits superior antimicrobial activity and more pronounced plaque biofilm disruption than QASeV when combined with light irradiation (MVL-210 photoreactor, 350-600 nm, 50 mW/cm2), and a better effect on reducing the diversity and restoring the structure of subgingival flora in periodontitis rat model was found through 16S rRNA gene sequencing analysis. The histological and Micro-CT analyses reveal that HASeV-based aPDT has a better therapeutic effect in reducing periodontal tissue inflammation and alveolar bone resorption. This work provides a new strategy for the development of viologen-based photosensitizers, which may be a favorable candidate for the aPDT against periodontitis.

Corrigendum: Enhanced immunogenicity and protective efficacy in mice following a Zika DNA vaccine designed by modulation of membrane-anchoring regions and its association to adjuvants.

[This corrects the article DOI: 10.3389/fimmu.2024.1307546.].

Enhanced immunogenicity and protective efficacy in mice following a Zika DNA vaccine designed by modulation of membrane-anchoring regions and its association to adjuvants.

Zika virus (ZIKV) is a re-emerging pathogen with high morbidity associated to congenital infection. Despite the scientific advances since the last outbreak in the Americas, there are no approved specific treatment or vaccines. As the development of an effective prophylactic approach remains unaddressed, DNA vaccines surge as a powerful and attractive candidate due to the efficacy of sequence optimization in achieving strong immune response. In this study, we developed four DNA vaccine constructs encoding the ZIKV prM/M (pre-membrane/membrane) and E (envelope) proteins in conjunction with molecular adjuvants. The DNA vaccine candidate (called ZK_ΔSTP), where the entire membrane-anchoring regions were completely removed, was far more immunogenic compared to their counterparts. Furthermore, inclusion of the tPA-SP leader sequence led to high expression and secretion of the target vaccine antigens, therefore contributing to adequate B cell stimulation. The ZK_ΔSTP vaccine induced high cellular and humoral response in C57BL/6 adult mice, which included high neutralizing antibody titers and the generation of germinal center B cells. Administration of ZK-ΔSTP incorporating aluminum hydroxide (Alum) adjuvant led to sustained neutralizing response. In consistency with the high and long-term protective response, ZK_ΔSTP+Alum protected adult mice upon viral challenge. Collectively, the ZK_ΔSTP+Alum vaccine formulation advances the understanding of the requirements for a successful and protective vaccine against flaviviruses and is worthy of further translational studies.

Organo-Ptii Complexes for Potent Photodynamic Inactivation of Multi-Drug Resistant Bacteria and the Influence of Configuration.

PtII based organometallic photosensitizers (PSs) have emerged as novel potent photodynamic inactivation (PDI) reagents through their enhanced intersystem crossing (ISC) processes. Currently, few PtII PSs have been investigated as antibacterial materials, with relatively poor performances reported and with structure-activity relationships not well described. Herein, a pair of configurational isomers are reported of Bis-BODIPY (4,4-difluoro-boradizaindacene) embedded PtII PSs. The cis-isomer (cis-BBP) displayed enhanced 1O2 generation and better bacterial membrane anchoring capability as compared to the trans-isomer (trans-BBP). The effective PDI concentrations (efficiency > 99.9%) for cis-BBP in Acinetobacter baumannii (multi-drug resistant (MDR)) and Staphylococcus aureus are 400 nM (12 J cm-2) and 100 nM (18 J cm-2), respectively; corresponding concentrations and light doses for trans-BBP in the two bacteria are 2.50 µM (30 J cm-2) and 1.50 µM (18 J cm-2), respectively. The 50% and 90% minimum inhibitory concentration (MIC50 and MIC90) ratio of trans-BBP to cis-BBP is 22.22 and 24.02 in A. baumannii (MDR); 21.29 and 22.36 in methicillin resistant S. aureus (MRSA), respectively. Furthermore, cis-BBP displays superior in vivo antibacterial performance, with acceptable dark and photoinduced cytotoxicity. These results demonstrate cis-BBP is a robust light-assisted antibacterial reagent at sub-micromolecular concentrations. More importantly, configuration of PtII PSs should be an important issue to be considered in further PDI reagents design.

Generation of a Hydrophobic Protrusion on Nanoparticles to Improve the Membrane-Anchoring Ability and Cellular Internalization.

Controlling the nanoparticle-cell membrane interaction to achieve easy and fast membrane anchoring and cellular internalization is of great importance in a variety of biomedical applications. Here we report a simple and versatile strategy to maneuver the nanoparticle-cell membrane interaction by creating a tunable hydrophobic protrusion on Janus particles through swelling-induced symmetry breaking. When the Janus particle contacts cell membrane, the protrusion will induce membrane wrapping, leading the particles to docking to the membrane, followed by drawing the whole particles into the cell. The efficiencies of both membrane anchoring and cellular internalization can be promoted by optimizing the size of the protrusion. In vitro, the Janus particles can quickly anchor to the cell membrane in 1 h and be internalized within 24 h, regardless of the types of cells involved. In vivo, the Janus particles can effectively anchor to the brain and skin tissues to provide a high retention in these tissues after intracerebroventricular, intrahippocampal, or subcutaneous injection. This strategy involving the creation of a hydrophobic protrusion on Janus particles to tune the cell-membrane interaction holds great potential in nanoparticle-based biomedical applications.

Cell Membrane-Anchored SERS Biosensor for the Monitoring of Cell-Secreted MMP-9 during Cell-Cell Communication.

Matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme, degrades the extracellular matrix and plays a key role in cell communication. However, the real-time monitoring of cell-secreted MMP-9 during cell-cell communication remains a challenge. Herein, we developed a cell-based membrane-anchored surface-enhanced Raman scattering (SERS) biosensor using a Au@4-mercaptobenzonitrile (4-MBN) @Ag@peptide nanoprobe for the monitoring of cell-secreted MMP-9 during cell communication. The multifunctional nanoprobe was created with Au@4-MBN@Ag acting as an interference-free SERS substrate with high enhancement in which the peptide not only serves to anchor the cell membrane but also provides MMP-9-activatable cleaved peptide chains. MMP-9-mediated cleavage resulted in the detachment of the Au@4-MBN@Ag nanoparticles from the cell membrane, thereby decreasing the SERS signals of cancer cells. The cell membrane-anchored SERS biosensor enables the real-time monitoring of cell-secreted MMP-9 during the interaction of MCF-7 and HUVEC cells. This study successfully demonstrates the dynamic change of cell-secreted MMP-9 during the communication between MCF-7 cells and HUVEC cells. The proposed nanoprobe was also utilized to precisely evaluate the breast and hepatoma cancer cell aggressiveness. This study provides a novel strategy for real-time monitoring of MMP-9 secretion during cell communication, which is promising for the investigation of the mechanisms underlying different tumor processes.

Dual-Pronged Attack: pH-Driven Membrane-Anchored NIR Dual-Type Nano-Photosensitizer Excites Immunogenic Pyroptosis and Sequester Immune Checkpoint for Enhanced Prostate Cancer Photo-Immunotherapy.

Prostate cancer (PCa) is a frustrating immunogenic "cold" tumor and generally receives unsatisfied immunotherapy outcomes in the clinic. Pyroptosis is an excellent immunogenic cell death form that can effectively activate the antitumor immune response, promote cytotoxic T-lymphocyte infiltration, and convert tumors from "cold" to "hot." However, the in vivo application of pyroptosis drugs is seriously limited, and the upregulation of tumor PD-L1 caused by photo-immunotherapy further promotes immune escape. Herein, a new nano-photosensitizer (YBS-BMS NPs-RKC) with pH-response integrating immunogenic pyroptosis induction and immune checkpoint blockade is developed. The pH-responsive polymer equipped with the cell membrane anchoring peptide RKC is used as the carrier and further encapsulated with the near-infrared-activated semiconductor polymer photosensitizer YBS and a PD-1/PD-L1 complex small molecule inhibitor BMS-202. The pH-driven membrane-anchoring and pyroptosis activation of YBS-BMS NPs-RKC is clearly demonstrated. In vitro and in vivo studies have shown that this dual-pronged therapy stimulates a powerful antitumor immune response to suppress primary tumor progression and evokes long-term immune memory to inhibit tumor relapse and metastasis. This work provides an effective self-synergistic platform for PCa immunotherapy and a new idea for developing more biocompatible photo-controlled pyroptosis inducers.

New-generation cytopharmaceuticals with powerfully boosted extravasation for enhanced cancer therapy.

Effective extravasation of therapeutic agents into solid tumors still faces huge challenges. Since the doubted effectiveness of enhanced penetration and retention effect, first-generation neutrophil cytopharmaceuticals with encapsulated drugs have been developed to improve the drug accumulation in tumors based on the active chemotaxis and extravasation of neutrophils. Herein, a new generation of neutrophil cytopharmaceuticals with enhanced tumor-specific extravasation is reported to satisfy more complex clinical demands. This neutrophil cytopharmaceutical is obtained by anchoring vascular endothelial growth factor receptor 2 (VEGFR2)-targeting peptide K237 on neutrophil membrane after endocytosis of chemotherapeutics by neutrophils. Leveraging the cytokine-mediated active migration of neutrophils, the specific-recognition of K237 peptide to tumor vascular endothelium expedites the migration and enhances tight adhesion of neutrophils to vascular endothelium, thus improving the extravasation of therapeutic agents to target sites. Moreover, anti-angiogenesis effect from VEGFR2-blocking by K237 peptide achieves a cooperative tumor destruction with cytotoxic effects from released chemotherapeutics. This study demonstrates the great potential of enhanced proactive extravasation of cytopharmaceuticals via a cell-anchoring technology, leading to expedited drug infiltration and boosted therapeutic effects, which can be applied in other cell therapies to improve efficacy.

Multi-Catcher Polymers Regulate the Nucleolin Cluster on the Cell Surface for Cancer Therapy.

Cell signal transduction mediated by cell surface ligand-receptor is crucial for regulating cell behavior. The oligomerization or hetero-aggregation of the membrane receptor driven by the ligand realizes the rearrangement of apoptotic signals, providing a new ideal tool for tumor therapy. However, the construction of a stable model of cytomembrane receptor aggregation and the development of a universal anti-tumor therapy model on the cellular surface remain challenging. This work describes the construction of a "multi-catcher" flexible structure GC-chol-apt-cDNA with a suitable integration of the oligonucleotide aptamer (apt) and cholesterol (chol) on a polymer skeleton glycol chitosan (GC), for the regulation of the nucleolin cluster through strong polyvalent binding and hydrophobic membrane anchoring on the cell surface. This oligonucleotide aptamer shows nearly 100-fold higher affinity than that of the monovalent aptamer and achieves stable anchoring to the plasma membrane for up to 6 h. Moreover, it exerts a high tumor inhibition both in vitro and in vivo by activating endogenous mitochondrial apoptosis pathway through the cluster of nucleolins on the cell membrane. This multi-catcher nano-platform combines the spatial location regulation of cytomembrane receptors with the intracellular apoptotic signaling cascade and represents a promising strategy for antitumor therapy.

Membrane Anchorage-Induced (MAGIC) Knockdown of Non-synonymous Point Mutations.

The promise of personalized medicine for monogenic and complex polygenic diseases depends on the availability of strategies for targeted inhibition of disease-associated polymorphic protein variants. Loss of function variants, including non-synonymous single nucleotide variants (nsSNVs) and insertion/deletion producing a frameshift, account for the vast majority of disease-related genetic changes. Because it is challenging to interpret the functional consequences of nsSNVs, they are considered a big barrier for personalized medicine. A method for inhibiting the specific expression of nsSNVs without editing the human genome will facilitate the elucidation of the biology of nsSNVs, but such a method is currently lacking. Here, I describe the phenomenon of membrane anchorage-induced (MAGIC) knockdown of allele-specific inhibition of protein and mRNA expression upon inner membrane tethering of point mutation-specific monoclonal antibodies (mAb). This phenomenon is likely mediated by a mechanism distinct from the protein degradation pathways, as the epitope-specific knockdown is replicated upon intracellular expression of a membrane-anchored single domain intrabody that lacks the Fc domain of the mAb. By harnessing the MAGIC knockdown of epitope-containing protein targets, I report a novel approach for inhibiting the expression of amino-acid-altering germline and somatic nsSNVs. As a proof-of-concept, I show the inhibition of human disease-associated variants namely, FGFR4 p.G388R, KRAS p.G12D and BRAF p.V600E protein variants. This method opens up a new avenue for not just therapeutic suppression of undruggable protein variants, but also for functional interrogation of the nsSNVs of unknown significance.

Systematic Interrogation of Cellular Signaling in Live Cells Using a Membrane-Anchored DNA Multitasking Processor.

Systematic interrogation of correlative signaling components in their native environment is of great interest for dissecting sophisticated cellular signaling. However, it remains a challenge because of the lack of versatile and effective approaches. Herein, we propose a cell membrane-anchored DNA multitasking processor acting as a "traffic light" for integrated analyses of cellular signal transduction. Enhanced and controllable inhibition of c-Met signaling was achieved by membrane-anchoring of DNA processors. Moreover, the multitasking capability of the DNA processor allowed the monitoring of correlative VEGF secretion induced by c-Met activity regulation directly. By exploiting versatile aptameric nucleic acids, this modular designed DNA multitasking processor dissected how cell surface receptors coordinated with related components in live cells systematically. Therefore, it provides a powerful chemical tool for both fundamental cell biology research and precision medicine applications.

A tumor acidity-driven transformable polymeric nanoassembly with deep tumor penetration and membrane-anchoring capability for targeted photodynamic therapy.

In recent years, directly damaging cell membrane therapeutic modalities have attracted great attention in the field of cancer therapy due to their critical role in guaranteeing essential cellular function. In this study, the transformable nanoassembly PEG-Ce6@PAEMA, consisting of the photosensitizer polyethylene glycol-chlorin-e6 (PEG-Ce6) and tumor pH-sensitive polymer poly(2-azepane ethyl methacrylate) (PAEMA), was developed for highly efficient membrane-targeted photodynamic therapy. The PAEMA core is rapidly protonated at the acidic tumor pH, resulting in the disassembly of PEG-Ce6@PAEMA and regeneration of PEG-Ce6. Subsequently, the resultant PEG-Ce6 with a very small size (~2.6 kDa) ensures deep penetration into tumor tissue and direct and rapid anchoring to the cancer cell membrane, eventually achieving superior tumor growth inhibition under light irradiation. Thus, this tumor acidity-driven transformable polymeric nanoassembly provides a simple but efficient strategy for membrane targeting cancer therapy.

Activation of Pyroptosis by Membrane-Anchoring AIE Photosensitizer Design: New Prospect for Photodynamic Cancer Cell Ablation.

Pyroptosis as a lytic and inflammatory form of cell death is a powerful tool to fight against cancer. However, pyroptosis is usually activated by chemotherapeutic drugs, which limits its anti-tumor applications due to drug resistance and severe side effects. Herein, we demonstrate that membrane targeting photosensitizers can induce pyroptosis for cancer cell ablation with noninvasiveness and low side effects. A series of membrane anchoring photosensitizers (TBD-R PSs) with aggregation-induced emission (AIE) characteristics were prepared through conjugation of TBD and phenyl ring with cationic chains. Upon light irradiation, cytotoxic ROS were produced in situ, resulting in direct membrane damage and superior cancer cell ablation. Detailed study revealed that pyroptosis gradually became the dominant cell death pathway along with the increase of TBD-R PSs membrane anchoring capability. This study offers a photo-activated pyroptosis-based intervention strategy for cancer cell ablation.

Engineering and Rewiring of a Calcium-Dependent Signaling Pathway.

An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes. Calcium-dependent regulation is an attractive approach for achieving such control, as diverse stimuli induce calcium influx by activating membrane channel receptors. Most calcium-dependent gene circuits use the endogenous nuclear factor of activated T-cells (NFAT) signaling pathway. Here, we employed engineered NFAT transcription factors to induce the potent and robust activation of exogenous gene expression in HEK293T cells. Furthermore, we designed a calcium-dependent transcription factor that does not interfere with NFAT-regulated promoters and potently activates transcription in several mammalian cell types. Additionally, we demonstrate that coupling the circuit to a calcium-selective ion channel resulted in capsaicin- and temperature-controlled gene expression. This engineered calcium-dependent signaling pathway enables tightly controlled regulation of gene expression through different stimuli in mammalian cells and is versatile, adaptable, and useful for a wide range of therapeutic and diagnostic applications.

Aromatic interactions with membrane modulate human BK channel activation.

Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue 'C-linker'. To determine the linker's role in human BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

Development and functional analysis of an anticancer T-cell medicine with immune checkpoint inhibitory ability.

Adoptive cell therapy using patients' own T-cells is expected to be an ideal cancer treatment strategy with excellent antitumor effects and low side effects. However, this therapy targeting solid tumors is unlikely to be effective because tumor tissues have an environment that suppresses T-cell function. In particular, interaction between programmed death-1 (PD-1) and its ligand (PD-L1) inhibits T-cell activation by which T-cells eliminate tumor cells. Here, we attempted to develop T-cells that can exert potent antitumor activity even in tumor tissues by genetically modifying them to express the anti-PD-L1 membrane-anchoring type single chain variable fragment (M-scFv) that can inhibit PD-L1/PD-1 interaction. Anti-PD-L1 M-scFv could be expressed on T-cells while maintaining PD-L1-binding ability. Although T-cell proliferation induced by CD3 stimulation was decreased depending on the PD-L1 stimulation intensity, M-scFv-expressing T-cells showed high proliferative activity even in the presence of PD-L1 by avoiding the PD-L1/PD-1-mediated suppression. Furthermore, M-scFv-expressing T-cells showed higher cytotoxic activity against PD-L1high tumor cells than that of mock T-cells. The effect of PD-L1/PD-1 blockade was more pronounced when the therapeutic target was low-antigenic tumor cells with low major histocompatibility complex expression, presenting only the shared antigen. These results indicated that anti-PD-L1 M-scFv expression was functional in avoiding T-cell dysfunction by PD-L1/PD-1 interaction. Our concept of anti-PD-L1 M-scFv-expressing T-cells is thus expected to improve the efficacy of T-cell therapy and contribute to simplify the treatment system and reduce treatment costs compared with the combination therapy of T-cells and antibodies.

Expression of cholinesterases and their anchoring proteins in rat heart.

Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia-reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.

Structural insight into the mechanism of action of antimicrobial peptide BMAP-28(1-18) and its analogue mutBMAP18.

Structural characterization of BMAP-28(1-18), a potent bovine myeloid antimicrobial peptide can aid in understanding its mechanism of action at molecular level. We report NMR structure of the BMAP-28(1-18) and its mutated analogue mutBMAP18 in SDS micelles. Structural comparison of the peptides bound to SDS micelles and POPE-POPG vesicles using circular dichroism, suggest that structures in the two lipid preparations are similar. Antimicrobial assays show that even though both these peptides adopt helical conformation, BMAP-28(1-18) is more potent than mutBMAP18 in killing bacterial cells. Our EM images clearly indicate that the peptides target the bacterial cell membrane resulting in leakage of its contents. The structural basis for difference in activity between these peptides was investigated by molecular dynamics simulations. Inability of the mutBMAP18 to retain its helical structure in presence of POPE:POPG membrane as opposed to the BMAP-28(1-18) at identical peptide/lipid ratios could be responsible for its decreased activity. Residues Ser5, Arg8 and Arg12 of the BMAP-28(1-18) are crucial for its initial anchoring to the bilayer. We conclude that along with amphipathicity, a stable secondary structure that can promote/initiate membrane anchoring is key in determining membrane destabilization potential of these AMPs. Our findings are a step towards understanding the role of specific residues in antimicrobial activity of BMAP-28(1-18), which will facilitate design of smaller, cost-effective therapeutics and would also help prediction algorithms to expedite screening out variants of the parent peptide with greater accuracy.

Yeast Mitoribosome Large Subunit Assembly Proceeds by Hierarchical Incorporation of Protein Clusters and Modules on the Inner Membrane.

Mitoribosomes are specialized for the synthesis of hydrophobic membrane proteins encoded by mtDNA, all essential for oxidative phosphorylation. Despite their linkage to human mitochondrial diseases and the recent cryoelectron microscopy reconstruction of yeast and mammalian mitoribosomes, how they are assembled remains obscure. Here, we dissected the yeast mitoribosome large subunit (mtLSU) assembly process by systematic genomic deletion of 44 mtLSU proteins (MRPs). Analysis of the strain collection unveiled 37 proteins essential for functional mtLSU assembly, three of which are critical for mtLSU 21S rRNA stability. Hierarchical cluster analysis of mtLSU subassemblies accumulated in mutant strains revealed co-operative assembly of protein sets forming structural clusters and preassembled modules. It also indicated crucial roles for mitochondrion-specific membrane-binding MRPs in anchoring newly transcribed 21S rRNA to the inner membrane, where assembly proceeds. Our results define the yeast mtLSU assembly landscape in vivo and provide a foundation for studies of mitoribosome assembly across evolution.