Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: Domain architecture of human Drg and Dfrp proteins. A. Human Drg GTPase containing an N-terminal helix-turn-helix (HTH) domain, a central G-domain with an S5D2L insertion, and a C-terminal TGS domain. The TGS domain is essential for binding to Dfrp1 and Dfrp2 and association with the translating ribosome. Drg1 and Drg2 share significant sequence homology with each other. Both HTH and TGS domains are essential for Drg1’s association with the microtubule. Drg1 and Drg2 lack a mitochondrial targeting signal at the N-terminus, suggesting they may not localize into mitochondria. Additionally, Drg1/2 do not share any sequence homology to known lipid-binding domains of mitochondrial outer membrane proteins, as shown in the top panel indicated by the red dashed line. B. Domain architecture of human Dfrp proteins. Dfrp1 has two zinc finger domains Like Drg, Dfrp lacks a mitochondrial targeting signal. Dfrp1 shows no sequence homology to known lipid-binding domains of mitochondrial outer membrane proteins, as shown in the top panel indicated by the red dashed line.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Binding Assay, Sequencing, Membrane

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: A. Immunofluorescence imaging of Drg1. Nuclei were stained with DAPI (blue), mitochondria with MitoTracker (red), and Drg1/Dfrp1 with antibodies (green, as indicated). Drg1 proteins predominantly localize to the cytoplasm, with a fraction colocalizing with mitochondria. B. Presence of Drg1 and Dfrp1 in both cytoplasmic and mitochondrial fractions. Western blot analysis of subcellular fractions confirming the presence of Drg1 and Dfrp1 in both cytoplasmic (denoted as “C”) and mitochondrial (denoted as “M”) fractions. C. Controlled protease protection assay using Proteinase K on purified mitochondria, demonstrating that Drg1 and Dfrp1 are localized to the outer mitochondrial membrane (OMM).

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Immunofluorescence, Imaging, Staining, Western Blot, Purification, Membrane

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: Endogenous Dfrp1 proteins localize in the cytoplasm Immunofluorescence imaging showing localization of Dfrp1 in ΔDrg1 HEK293T cells. Nuclei were stained with DAPI (blue), mitochondria with MitoTracker (red), and Dfrp1 with antibodies (green). Almost all Dfrp1 localizes to the cytoplasm, with a fraction colocalizing to the mitochondrial periphery.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Immunofluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: Drg1 associates with cytoplasmic ribosomes at the mitochondrial outer membrane. A. Immunofluorescence microscopy showing Drg1 (green) and cytoplasmic ribosomes (shown via its marker Rps6, purple) with partial colocalization at mitochondria (MitoTracker, red); nuclei are stained with DAPI (blue). Both Drg1 and cytoplasmic ribosomes exhibit enrichment at the mitochondrial periphery. B. Western blot analysis of cytoplasmic (C) and mitochondrial (M) fractions confirms the presence of both Drg1 and cytoplasmic ribosomes in the mitochondrial fraction. Ribosomes were detected via Rps6 marker. Note that deletion of cellular Drg1 has little effect on the presence of ribosomes. Drg1 localization at the mitochondrial OMM depends on its own expression. C. The TGS domain of Drg1 is required for its association with cytoplasmic ribosomes at the mitochondrial outer membrane. Full-length Drg1 and ΔHTH_Drg1 localize to the mitochondrial fraction in ΔDrg1 HEK293T cells, whereas ΔTGS_Drg1 fails to do so. Because the TGS domain is specifically required for ribosome and Dfrp1 binding, but not microtubule association, these results indicate that Drg1 localizes to mitochondria through interaction with ribosome-associated complexes rather than microtubules.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Membrane, Immunofluorescence, Microscopy, Marker, Staining, Western Blot, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: Drg1 and ribosomal localization at mitochondria, and domain requirements for mitochondrial targeting. A. Endogenous Drg1 and RPS6 (cytoplasmic ribosome marker) colocalize at the mitochondrial outer membrane. HEK293T cells were immunostained with anti-Drg1 (green) and anti-RPS6 (violet) antibodies, with MitoTracker labeling mitochondria (red) and DAPI labeling nuclei (blue). Confocal microscopy shows both Drg1 and RPS6 localize to mitochondria, suggesting ribosomal association with Drg1 at the OMM. B. Drg1 domain requirements for mitochondrial localization. ΔDrg1 HEK293T cells expressing Drg1 mutants (ΔHTH or ΔTGS) were imaged as in panel A. Deletion of the TGS domain abolishes mitochondrial localization, whereas ΔHTH retains localization. These results indicate the TGS domain is critical for Drg1 targeting to mitochondria and/or binding to Dfrp1 and ribosomes.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Marker, Membrane, Labeling, Confocal Microscopy, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: Loss of Drg1 alters mitochondrial morphology and impairs mitochondrial function. (A-C) Loss of Drg1 affects mitochondrial morphology. A. Mitochondria in ΔDrg1 cells are more oval and swollen compared to wild-type (WT) cells, as shown via fluorescence confocal imaging of mitochondria (red, MitoTracker). B. Mitochondria in ΔDrg1 cells are larger than those in WT cells. Using confocal imaging, approximately 8000 mitochondria from WT and ΔDrg1 cells were assessed. Quantification of mitochondrial area in WT and ΔDrg1 cells was done using the FIJI plugin, which indicates that ΔDrg1 cells have an average area of 0.4 µm², compared to 0.2 µm² in WT cells. C. An increased fraction of “swollen” mitochondria in ΔDrg1 cells. Using the average mitochondrial area in WT cells as a benchmark, over 60% of mitochondria in ΔDrg1 cells are swollen, compared to ∼35% in WT cells. D. Reduced mitochondrial membrane potential in ΔDrg1 cells. Reduced mitochondrial membrane potential in ΔDrg1 cells compared to the WT, measured by TMRM staining (in green) and fluorescence confocal microscopy imaging.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Fluorescence, Imaging, Membrane, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: ΔDrg1 cells exhibit increased mitochondrial fragmentation and swelling. Confocal fluorescence imaging of WT and ΔDrg1 HEK293T cells showing ΔDrg1 cells have more fragmented, swollen, and round mitochondria compared to WT HEK293T cells. Nuclei were stained with DAPI (blue) and mitochondria with MitoTracker (red). Representative images demonstrate that ΔDrg1 cells (Left panel) exhibit more swollen mitochondria compared to WT cells (Right panel).

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Fluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: Loss of Drg1 impairs mitochondrial protein import. A. Schematic of a mitochondrial import reporter consisting of the SOD2 mitochondrial targeting sequence (MTS) and SOD2 3′UTR flanking an eGFP coding region. SOD2 is a nuclear-encoded mitochondrial enzyme (mitochondrial peroxidase), and the MTS/3′UTR elements promote delivery of newly synthesized eGFP to mitochondria after transfection into HEK293T cells. Import efficiency is quantified by eGFP fluorescence. B. Drg1 loss compromises mitochondrial protein import. (i) Localization of the eGFP mRNA to the mitochondria. The DNA construct shown in (A) was cloned and transfected into HEK293T cells. Following mitochondrial and cytosolic fractionation, translating ribosomes were pelleted and mRNA was extracted. qPCR analysis confirms the presence of construct mRNA in the mitochondrial polysome fraction. (ii). Functional eGFP proteins are only found in mitochondria. HEK293T cells were transfected with the plasmid containing the cloned DNA construct. Fluorescence confocal imaging reveals eGFP (green) localizing to mitochondria that were labeled with MitoTracker (red). Nuclei were counterstained with DAPI (blue). (iii) A reduced median of eGFP fluorescence signal in ΔDrg1 cells. A reduced median of eGFP fluorescence signal in ΔDrg1 cells expressing the reporter compared to WT cells, as measured by flow cytometry. This observation is consistent with decreased mitochondrial import capacity in the absence of Drg1. B. Loss of Drg1 alone does not induce apoptosis. MTT assay measuring cellular viability and proliferation shows no significant difference between WT and ΔDrg1 cells despite changes in mitochondrial morphology and function.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Sequencing, Synthesized, Transfection, Fluorescence, Construct, Clone Assay, Fractionation, Functional Assay, Plasmid Preparation, Imaging, Labeling, Expressing, Flow Cytometry, MTT Assay

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: APEX2-Drg1 experimental workflow and data quality. A. Schematic of the Drg1-APEX2 proximity labeling workflow, including APEX2-catalyzed biotinylation, subcellular fractionation, streptavidin pulldown, and LC-MS/MS analysis. B. Hierarchical clustering heatmap of Drg1-APEX2 LC-MS/MS data from three biological replicates. Replicates cluster together within each condition, confirming reproducibility and high data quality. AD-P denotes cytosolic polysome samples, and AD-M denotes mitochondrial samples.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Labeling, Fractionation, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: Drg1 interactome at the mitochondrial outer membrane in HEK293T human cells. A. Proximity labeling identifies Drg1-associated proteins at mitochondria. HEK293T cells expressing Drg1-APEX2 underwent proximity-dependent biotinylation. Biotinylated proteins were enriched from the mitochondrial fraction and the 80S cytoplasmic ribosome fraction using streptavidin pulldown and identified by LC-MS/MS. The volcano plot displays significantly enriched proteins in the mitochondrial fraction (log 2 fold change >1, Benjamini-Hochberg adjusted p < 0.05). Proteins known to undergo co-translational mitochondrial import are labeled. Proteins are colored by annotated subcellular localization (Uniprot): cytosol, membrane/ER/Golgi, mitochondrion, nucleus, or other. A subset of identified proteins is shown with their corresponding amino acid (AA) length. All these proteins were reported to undergo co-translational import into mitochondria at the outer mitochondrial membrane, except for NUCB2 (*), which is a protein primarily associated with the endoplasmic reticulum (ER). B. Gene Ontology enrichment analysis of mitochondrial Drg1-interacting proteins, showing terms for cellular compartment, biological process, and molecular function.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Membrane, Labeling, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet:

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Labeling, Molecular Weight, Sequencing

Journal: bioRxiv

Article Title: Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

doi: 10.64898/2026.02.23.707305

Figure Lengend Snippet: Loss of Drg1 compromises protein homeostasis A. Loss of Drg1 alters ribosome occupancy of mitochondrial protein–encoding mRNAs without changing total mRNA abundance. Total cellular mRNA and ribosome-protected (translating) mRNA were purified from WT and ΔDrg1 HEK293T cells, converted into cDNA libraries, and analyzed by qPCR. Transcripts encoding mitochondrial ribosomal proteins involved in mitochondrial translation (MRPS30 and MRPL43) and ATP5D, a subunit of the electron transport chain complex V required for ATP production, were examined. GAPDH served as a normalization control. Ct values for GAPDH were comparable between WT and ΔDrg1 cells in both total and translating mRNA pools, indicating equivalent input and fractionation efficiency. No significant differences were detected in total mRNA levels of MRPS30, MRPL43, or ATP5D between WT and ΔDrg1 cells (data not shown). In contrast, ribosome occupancy— calculated as the ratio of transcript abundance in the translating fraction to that in the total mRNA fraction—was substantially altered for each gene in ΔDrg1 cells, indicating impaired translational engagement of mitochondria-related mRNAs upon loss of Drg1. B. Loss of Drg1 alters the mRNA abundance of genes involved in mitochondrial fusion and fission, as quantified by qPCR.

Article Snippet: HEK293T cells were grown and transfected with plasmid expressing N-terminal V5-APEX-10AA linker tagged Drg1 gene with Transit (MIR2705, MirusBio) and serum free media (Gibco) at ∼70-80% confluency.

Techniques: Purification, Control, Fractionation

Journal: Journal of Virology

Article Title: A recurrent adaptive mutation in the transmembrane 2B protein of an insect picorna-like virus in a nonnative host

doi: 10.1128/jvi.01239-25

Figure Lengend Snippet: Sting colocalizes with the CrPV 2B protein. ( A ) Confocal microscopy images of Drosophila S2 cells transfected with a combination of the indicated plasmids. Cells were fixed 48 h after transfection and stained with antibodies against the V5 tag and the cis Golgi protein GM130. ( B ) Violin plot of Pearson’s correlation coefficient (R) of Sting with eGFP ( n = 27), 2B-eGFP ( n = 22), or 2B(D29N)-eGFP ( n = 22) in individual cells, calculated in Fiji using the Costes method. The median is represented with a solid black line and the quartiles with dashed lines. Adjusted P -values from Tukey’s multiple comparison test are indicated with asterisks: ** P < 0.01, *** P < 0.001. ( C ) Co-immunoprecipitation of Sting with 2B and 2B(D29N). S2 cells were transfected with the indicated plasmids, and the cell lysates (input) and eGFP immunoprecipitates (eGFP ImmP) were analyzed by western blot using eGFP, V5, and GAPDH antibodies. The white arrow heads indicate Sting-V5 (expected size 43.1 kDa), black arrow heads indicate 2B-eGFP and 2B(D29N)-eGFP (43.3 kDa), and green arrow heads indicate eGFP. As previously reported for human STING, Drosophila Sting showed distinct migrating bands, which might be due to post-translational modifications . M, size marker.

Article Snippet: Membranes were stained with antibodies against GFP (rat, 1:1000, ChromoTek, 3h9-150), V5 tag (mouse, 1:2000, Invitrogen, 46-0705), or GAPDH (rabbit, 1:1000, Bioss, bs-8778R), followed by secondary antibodies IRDye 800CW goat anti-rat IgG (LICORBio, 926-32219), IRDye 800CW goat anti-mouse IgG (LICORBio, 926-32210), and IRDye 800CW goat anti-rabbit IgG (LICORBio, 926-32211), respectively.

Techniques: Confocal Microscopy, Transfection, Staining, Comparison, Immunoprecipitation, Western Blot, Marker