pdelta 28e4 plasmid  (Addgene inc)

Journal: Journal of Virology

Article Title: The ACBD3 protein coordinates ER-Golgi contacts to enable productive TBEV infection

doi: 10.1128/jvi.02224-24

Figure Lengend Snippet: Identification of ACBD3 as a host factor in NS4B-APEX2 proximal protein analysis. ( A ) Confocal fluorescence micrographs of HEK293T cells transiently expressing TBEV NS4B-GFP infected with LGTV (MOI 10, 16 h.p.i.) and stained with anti-calnexin (CANX, ER marker) antibodies and anti-dsRNA antibodies. Scale bar, 10 µm. ( B ) Schematic illustration of the NS4B-APEX2 proximal protein biotinylation screen used in this study. ( C ) Volcano plots of the proteins identified in the NS4B-APEX2 proximal protein biotinylation screen in NS4B-APEX2 cells (left) and NS4B-APEX2 cells infected with LGTV (MOI 10, 16 h.p.i.) (right). Proteins are indicated by dots color-coded based on the FC and P -values. Three biological replicates per condition were analyzed, and the P -value was calculated using unpaired t -test. ( D ) Schematic illustration of ERES-Golgi contacts, and the top hit proteins ACBD3, TFG, SEC23IP, and KTN1 ( E ) Quantification of the percentage of GFP-positive KD HEK293T cells expressing E protein at 24 h after LGTV infection (MOI 1). The KD proteins are indicated. Mean ± SD of 3 independent experiments. One-way ANOVA with Dunnett multiple tests, *** P < 0.005, **** P < 0.0001.

Article Snippet: The coding sequence of APEX2 was PCR-amplified from APEX2 plasmid (a gift from Alice Ting, Addgene plasmid #72480, [ ]) using primers containing restriction sites NotI (5’) and XhoI (3’).

Techniques: Fluorescence, Expressing, Infection, Staining, Marker

Journal: bioRxiv

Article Title: STAR/STARD1: a mitochondrial intermembrane space cholesterol shuttle degraded through mitophagy

doi: 10.1101/2025.04.03.647084

Figure Lengend Snippet: ( A ) Lentiviral expression of a truncated STAR lacking its mitochondrial targeting sequence ( ΔN62- STAR) fused to the outer mitochondrial membrane (OMM) anchor TOM20 fails to rescue Bt 2 cAMP-induced steroidogenesis in STAR-deficient MA-10 STKO cells, in contrast to reconstitution with native STAR (positive control, pC; negative control, nC; different alphabets indicate significant difference p<0.05). Immunoblot confirms expression of the TOM20 -ΔN62- STAR fusion protein at the expected molecular weight (∼41.2 kDa). This contrasts with previous claims that this construct yields maximal constitutive steroid production. (B) Sequence comparisons between the STAR MTS and the well-characterized bipartite MTS of cytochrome C1 (CYTC1 MTS ) reveal conserved features, including a mitochondrial processing peptidase (MPP) cleavage site and a predicted inner membrane peptidase (IMP) site. These conserved elements support a stop-transfer import mechanism characteristic of proteins targeted to the mitochondrial intermembrane space (IMS). The accompanying schematic illustrates the principle of this stop-transfer mechanism, where translocases at the outer and inner mitochondrial membranes mediate import ①, and MPP-mediated cleavage ②, the sorting signal anchors to the IMM ③, followed by IMP-mediated cleavage ④, and IMS release ⑤. (C) Confocal imaging demonstrates mitochondrial colocalization of CYTC1 MTS-ΔN62- STAR-Emerald with the OMM marker TOM20-mRuby in MA-10 cells, confirming successful targeting to mitochondria using the CYTC1 MTS . (D) Functional rescue of steroidogenesis is achieved in MA-10 STKO cells by CYTC1 MTS-ΔN62- STAR, showing equivalence to native STAR in restoring progesterone production, thereby demonstrating that IMS localization fully restores STAR function (different alphabets indicate significant difference p<0.05). Western blot analysis shows identical of mitochondrial processing of native STAR and CYTC1 MTS-ΔN62- STAR. (E) APEX2-based ultrastructural labeling distinguishes STAR sub-mitochondrial localization: TOM20-APEX2 marks the cytoplasmic face of the OMM; N36- STAR-APEX2, containing only the presequence, targets the matrix; native STAR-APEX2 localizes specifically to the IMS. This confirms that the full STAR MTS directs import via a stop-transfer mechanism, releasing STAR into the IMS for its functional role.

Article Snippet: Fusions of STAR with an engineered ascorbate peroxidase (APEX2) for ultrastructural localization was cloned from mito-V5-APEX2 (72480; Addgene) downstream of native and N36-STAR in the pMX retroviral vector (pMX-STAR-APEX2, pMX-N36-APEX2).

Techniques: Expressing, Sequencing, Membrane, Positive Control, Negative Control, Western Blot, Molecular Weight, Construct, Imaging, Marker, Functional Assay, Labeling

Journal: bioRxiv

Article Title: STAR/STARD1: a mitochondrial intermembrane space cholesterol shuttle degraded through mitophagy

doi: 10.1101/2025.04.03.647084

Figure Lengend Snippet: ( A ) Lentiviral expression of a truncated STAR lacking its mitochondrial targeting sequence ( ΔN62- STAR) fused to the outer mitochondrial membrane (OMM) anchor TOM20 fails to rescue Bt 2 cAMP-induced steroidogenesis in STAR-deficient MA-10 STKO cells, in contrast to reconstitution with native STAR (positive control, pC; negative control, nC; different alphabets indicate significant difference p<0.05). Immunoblot confirms expression of the TOM20 -ΔN62- STAR fusion protein at the expected molecular weight (∼41.2 kDa). This contrasts with previous claims that this construct yields maximal constitutive steroid production. (B) Sequence comparisons between the STAR MTS and the well-characterized bipartite MTS of cytochrome C1 (CYTC1 MTS ) reveal conserved features, including a mitochondrial processing peptidase (MPP) cleavage site and a predicted inner membrane peptidase (IMP) site. These conserved elements support a stop-transfer import mechanism characteristic of proteins targeted to the mitochondrial intermembrane space (IMS). The accompanying schematic illustrates the principle of this stop-transfer mechanism, where translocases at the outer and inner mitochondrial membranes mediate import ①, and MPP-mediated cleavage ②, the sorting signal anchors to the IMM ③, followed by IMP-mediated cleavage ④, and IMS release ⑤. (C) Confocal imaging demonstrates mitochondrial colocalization of CYTC1 MTS-ΔN62- STAR-Emerald with the OMM marker TOM20-mRuby in MA-10 cells, confirming successful targeting to mitochondria using the CYTC1 MTS . (D) Functional rescue of steroidogenesis is achieved in MA-10 STKO cells by CYTC1 MTS-ΔN62- STAR, showing equivalence to native STAR in restoring progesterone production, thereby demonstrating that IMS localization fully restores STAR function (different alphabets indicate significant difference p<0.05). Western blot analysis shows identical of mitochondrial processing of native STAR and CYTC1 MTS-ΔN62- STAR. (E) APEX2-based ultrastructural labeling distinguishes STAR sub-mitochondrial localization: TOM20-APEX2 marks the cytoplasmic face of the OMM; N36- STAR-APEX2, containing only the presequence, targets the matrix; native STAR-APEX2 localizes specifically to the IMS. This confirms that the full STAR MTS directs import via a stop-transfer mechanism, releasing STAR into the IMS for its functional role.

Article Snippet: Fusions of STAR with an engineered ascorbate peroxidase (APEX2) for ultrastructural localization was cloned from mito-V5-APEX2 (72480; Addgene) downstream of native and N36-STAR in the pMX retroviral vector (pMX-STAR-APEX2, pMX-N36-APEX2).

Techniques: Expressing, Sequencing, Membrane, Positive Control, Negative Control, Western Blot, Molecular Weight, Construct, Imaging, Marker, Functional Assay, Labeling