Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Domain scheme of Erlin1 and Erlin2. b, Sequence alignment of human Erlin1 and Erlin2, with structural features noted above, colored according to domain. c, Coomassie stain (top) and immunoblotting (bottom) of the Erlin1/2 complex tandemly purified via N-terminally Strep-tagged Erlin1 and C-terminally Flag-tagged Erlin2. PD, pulldown; FT, flow-through; Elu, elution. d, Size exclusion chromatography (SEC) trace, showing absorbance at 280 nm values, of the tandem-purified Erlin1/2 complex as in c. The fractions corresponding to Erlin1/2 as indicated were pooled for cryo-EM analysis.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Sequencing, Staining, Western Blot, Purification, Size-exclusion Chromatography, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Model and secondary structural element organization of Erlin1 (left) and Erlin2 (right), colored by domain. b, Map-to-model fits for the indicated regions of Erlin1 (top) or Erlin2 (bottom).

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques:

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Surface representation of the Erlin1/2 complex model, with two individual subunits colored in pink and blue, and the other subunits colored according to the indicated domain. b, Surface representations of the Erlin1/2 complex model colored by molecular lipophilicity potential (mlp: teal – most hydrophilic, dark goldenrod – most lipophilic, left) or by Coulombic electrostatic potential (right). Orange and red arrowheads note hydrophobic and acidic surfaces. c, Model of the N-terminal transmembrane (TM) and SPFH1 domains of Erlin1 (pink) and Erlin2 (blue) with hydrophobic residues at the endoplasmic reticulum (ER) membrane interface indicated. Aromatic residues are indicated in bold. d, Model of alternating Erlin1 and Erlin2 subunits with the SPFH2 domains colored as in c. Residues involved in intersubunit hydrogen bonds are colored yellow and indicated. Residues that contribute to an acidic patch and N-linked glycosylation sites (N-glyc) are also indicated. e , Model of alternating Erlin and Erlin2 CC1 helices with hydrogen bonding interactions indicated. f, Model as in d with the positions of disease-linked mutations indicated in purple.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Membrane

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Clipped side view of the Erlin1/2 complex cryo-EM map with the elements that form four structural layers at the narrow end of the ‘cage’ colored as indicated. b, Top view of the Erlin1/2 complex model with the narrow end colored as in a. c, Isolated elements contributing to the four layers colored as in a. d, Hydrophobic packing and hydrogen bonding interactions involving the CC2 helices of Erlin that facilitate the transitions from the CC1 helices and into β9 of the CT. The CC2 helix of the ‘leftmost’ subunit, when viewed from the outside of the cage, is orange and modeled as Erlin2. The other two subunits are modeled as Erlin1 (pink) and Erlin2 (blue). e, Interactions involving the IPNMF motif in the Erlin CTs and that facilitate the turning of alternate Erlin CTs towards the outer edge of the cage. The leftmost subunit is modeled as Erlin2 and colored according to designations as in a. The other two subunits are modeled as Erlin1 (pink) and Erlin2 (blue). f, Positions of disease-linked mutations (purple) in the CC2 and CT domains of Erlin1 (pink) or Erlin2 (colored as in e or blue), viewed from the outside of the Erlin1/2 complex cage.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cryo-EM Sample Prep, Isolation

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Map-to-model fit of C-terminal PHB1 and PHB2 elements in the closed and open conformations. Note: PHB2 contains a C-terminal extension (dark blue) not present in PHB1. b, Close-up view of the hydrogen bonding network between the N-terminal portion of the CC1 helix and the β6-β7 loop in the SPFH2 domain of an adjacent subunit. c, Isolated elements contributing to the four layers at the narrow end of the PHB1/2 complex cage, colored as in . d, Side view of the cryo-EM map of the closed or open PHB1/2 complex, clipped near the center (top) shows a central channel (arrow) flanked by the PHB1 CT and the β9 ring (insets at bottom).

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Isolation, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Superposition of a PHB1/2 heterodimer in the closed (mauve and robin blue) and open (cream and gray) PHB1/2 complex conformations. b, Model of the PHB1/2 heterodimer in the closed complex conformation as in a, colored by root mean square deviation (RMSD) values compared to the open complex. Domain features are indicated. c, ‘Bottom’ view (top) of the closed and open PHB1/2 complex models with one heterodimer in each complex colored as in a and the others colored according to domain, and a rotated view showing only an isolated heterodimer (bottom). The rotation, relative to the centroid of the complex (top), and the translation (bottom) of the N-terminal residue of the PHB1 (K177, orange) or PHB2 (R191, purple) CC1 helix from the closed (mauve and robin blue) to open (cream and gray) conformation are indicated. d, Zoomed inset of the superposition as in a, showing the distance shifted by the indicated PHB1 (orange) or PHB2 (purple) residues in the SPFH domains between the closed and open conformations. e , Superposition of the isolated SPFH1 and SPFH2 domains of PHB1 and PHB2 in the closed and open PHB1/2 complex conformations, colored as in a.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cream, Isolation, Residue

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Surface representation of the closed (left) or open (right) PHB1/2 complex model, with two individual subunits colored as indicated, and the other subunits colored by domain. The buried area at the intersubunit interface on each side of the indicated PHB2 subunit is listed. b , Model of the N-terminal transmembrane (TM) and SPFH1 domains of PHB1 (mauve) and PHB2 (robin blue) with hydrophobic residues at the inner mitochondrial membrane (IMM) interface indicated. Aromatic residues are indicated in bold. IMS, intermembrane space. c, Model of alternating PHB1 and PHB2 subunits in the closed PHB1/2 complex with the SPFH2 domains colored as in a. Residues involved in intersubunit hydrogen bonds via their sidechain (yellow) or backbone (mauve or robin blue) are indicated. Residues involved in hydrophobic packing are orange. d, As in c, but for the open PHB1/2 complex and PHB1 colored cream. Residues indicated in bold maintain interactions in both the closed and open PHB1/2 complex. e, Model of alternating PHB1 and PHB2 CC1 helices in the closed (left) or open (right) complex, with hydrogen bonding interactions indicated as in c and d. f, Clipped side view of the cryo-EM map (left) or top view of the model (right) of the closed PHB1/2 complex with the elements that form four structural layers at the narrow end of the cage colored as indicated. g, Hydrogen bonding interactions involving the CC2 domains of PHB1 (mauve) and PHB2 (orange or robin blue), shown on the model of the closed PHB1/2 complex. The helix domain of the ‘leftmost’ PHB2 subunit, when viewed from the outside of the cage, is orange. h, Hydrophobic packing interactions involving the CC2 and CT domains of PHB1 (mauve) and PHB2 (colored according to the indicated 4-layer feature for the leftmost subunit, when viewed from the outside of the cage, or robin blue). Select residues of PHB1 (mauve), the PHB2 subunit to its left (orange), or the PHB2 subunit to its right (robin blue) are indicated.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Membrane, Cream, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Domain scheme of Erlin1 and Erlin2. b, Sequence alignment of human Erlin1 and Erlin2, with structural features noted above, colored according to domain. c, Coomassie stain (top) and immunoblotting (bottom) of the Erlin1/2 complex tandemly purified via N-terminally Strep-tagged Erlin1 and C-terminally Flag-tagged Erlin2. PD, pulldown; FT, flow-through; Elu, elution. d, Size exclusion chromatography (SEC) trace, showing absorbance at 280 nm values, of the tandem-purified Erlin1/2 complex as in c. The fractions corresponding to Erlin1/2 as indicated were pooled for cryo-EM analysis.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Sequencing, Staining, Western Blot, Purification, Size-exclusion Chromatography, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: Representative micrograph (top left) and cryo-EM processing pipeline for the Erlin1/2 complex, showing representative 2D classes and 3D volumes.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Cryo-EM map of the Erlin1/2 complex, with alternating subunits colored pink and blue, viewed from the ER lumen (“top”, left) and from the ER membrane (right). b, Side view of the cryo-EM map (left) and model of the Erlin1/2 complex superposed in a low-pass filtered map showing the detergent micelle (right) as a proxy for the ER membrane (dashed lines). The dimensions of the complex and placement of Erlin1/2 domains are indicated.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Model and secondary structural element organization of Erlin1 (left) and Erlin2 (right), colored by domain. b, Map-to-model fits for the indicated regions of Erlin1 (top) or Erlin2 (bottom).

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques:

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Surface representation of the Erlin1/2 complex model, with two individual subunits colored in pink and blue, and the other subunits colored according to the indicated domain. b, Surface representations of the Erlin1/2 complex model colored by molecular lipophilicity potential (mlp: teal – most hydrophilic, dark goldenrod – most lipophilic, left) or by Coulombic electrostatic potential (right). Orange and red arrowheads note hydrophobic and acidic surfaces. c, Model of the N-terminal transmembrane (TM) and SPFH1 domains of Erlin1 (pink) and Erlin2 (blue) with hydrophobic residues at the endoplasmic reticulum (ER) membrane interface indicated. Aromatic residues are indicated in bold. d, Model of alternating Erlin1 and Erlin2 subunits with the SPFH2 domains colored as in c. Residues involved in intersubunit hydrogen bonds are colored yellow and indicated. Residues that contribute to an acidic patch and N-linked glycosylation sites (N-glyc) are also indicated. e , Model of alternating Erlin and Erlin2 CC1 helices with hydrogen bonding interactions indicated. f, Model as in d with the positions of disease-linked mutations indicated in purple.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Membrane

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Clipped side view of the Erlin1/2 complex cryo-EM map with the elements that form four structural layers at the narrow end of the ‘cage’ colored as indicated. b, Top view of the Erlin1/2 complex model with the narrow end colored as in a. c, Isolated elements contributing to the four layers colored as in a. d, Hydrophobic packing and hydrogen bonding interactions involving the CC2 helices of Erlin that facilitate the transitions from the CC1 helices and into β9 of the CT. The CC2 helix of the ‘leftmost’ subunit, when viewed from the outside of the cage, is orange and modeled as Erlin2. The other two subunits are modeled as Erlin1 (pink) and Erlin2 (blue). e, Interactions involving the IPNMF motif in the Erlin CTs and that facilitate the turning of alternate Erlin CTs towards the outer edge of the cage. The leftmost subunit is modeled as Erlin2 and colored according to designations as in a. The other two subunits are modeled as Erlin1 (pink) and Erlin2 (blue). f, Positions of disease-linked mutations (purple) in the CC2 and CT domains of Erlin1 (pink) or Erlin2 (colored as in e or blue), viewed from the outside of the Erlin1/2 complex cage.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cryo-EM Sample Prep, Isolation

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Sidechains of the residues in parallel β9 strands, viewed from either the ‘outside’ or the center (‘inside’) of the Erlin1/2 complex cage. Note: the β9 sequences are identical, but the side that each sidechain resides alternates between adjacent Erlin subunits. b, Map-to-model fit of the C-terminal Erlin elements, viewed from the outside (top) or top (bottom) of the Erlin1/2 complex cage, with aromatic residues in β9, as in a, indicated. Note: clear divergence in the position of alternating CTs. c, Side view of the cryo-EM map of the Erlin1/2 complex, clipped near the center (top), shows a potential central channel (arrow) flanked by CT-in elements (inset at bottom). d, Top view of the Erlin1/2 complex model showing hydrophobic residues in the CT-in element (modeled as Erlin1) that flank a central channel-like structure.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Superposition of a PHB1/2 heterodimer in the closed (mauve and robin blue) and open (cream and gray) PHB1/2 complex conformations. b, Model of the PHB1/2 heterodimer in the closed complex conformation as in a, colored by root mean square deviation (RMSD) values compared to the open complex. Domain features are indicated. c, ‘Bottom’ view (top) of the closed and open PHB1/2 complex models with one heterodimer in each complex colored as in a and the others colored according to domain, and a rotated view showing only an isolated heterodimer (bottom). The rotation, relative to the centroid of the complex (top), and the translation (bottom) of the N-terminal residue of the PHB1 (K177, orange) or PHB2 (R191, purple) CC1 helix from the closed (mauve and robin blue) to open (cream and gray) conformation are indicated. d, Zoomed inset of the superposition as in a, showing the distance shifted by the indicated PHB1 (orange) or PHB2 (purple) residues in the SPFH domains between the closed and open conformations. e , Superposition of the isolated SPFH1 and SPFH2 domains of PHB1 and PHB2 in the closed and open PHB1/2 complex conformations, colored as in a.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cream, Isolation, Residue

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Surface representation of the closed (left) or open (right) PHB1/2 complex model, with two individual subunits colored as indicated, and the other subunits colored by domain. The buried area at the intersubunit interface on each side of the indicated PHB2 subunit is listed. b , Model of the N-terminal transmembrane (TM) and SPFH1 domains of PHB1 (mauve) and PHB2 (robin blue) with hydrophobic residues at the inner mitochondrial membrane (IMM) interface indicated. Aromatic residues are indicated in bold. IMS, intermembrane space. c, Model of alternating PHB1 and PHB2 subunits in the closed PHB1/2 complex with the SPFH2 domains colored as in a. Residues involved in intersubunit hydrogen bonds via their sidechain (yellow) or backbone (mauve or robin blue) are indicated. Residues involved in hydrophobic packing are orange. d, As in c, but for the open PHB1/2 complex and PHB1 colored cream. Residues indicated in bold maintain interactions in both the closed and open PHB1/2 complex. e, Model of alternating PHB1 and PHB2 CC1 helices in the closed (left) or open (right) complex, with hydrogen bonding interactions indicated as in c and d. f, Clipped side view of the cryo-EM map (left) or top view of the model (right) of the closed PHB1/2 complex with the elements that form four structural layers at the narrow end of the cage colored as indicated. g, Hydrogen bonding interactions involving the CC2 domains of PHB1 (mauve) and PHB2 (orange or robin blue), shown on the model of the closed PHB1/2 complex. The helix domain of the ‘leftmost’ PHB2 subunit, when viewed from the outside of the cage, is orange. h, Hydrophobic packing interactions involving the CC2 and CT domains of PHB1 (mauve) and PHB2 (colored according to the indicated 4-layer feature for the leftmost subunit, when viewed from the outside of the cage, or robin blue). Select residues of PHB1 (mauve), the PHB2 subunit to its left (orange), or the PHB2 subunit to its right (robin blue) are indicated.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Membrane, Cream, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: WDFY2 restrains matrix metalloprotease secretion and cell invasion by retention of VAMP3 in endosomal tubules

doi: 10.1101/299610

Figure Lengend Snippet: WDFY2 localization in the endocytic pathway. SIM image showing WDFY2 localization in relation to different markers of the early endocytic pathway. APPL1 (grey) was used as a marker for early vesicles and EEA1 (red) marks early endosome. GFP-WDFY2 (green) localizes to an independent vesicle pool (inset 2) and to subdomains on EEAl-labelled endosomes (inset 3). There is only limited overlap with APPL1 endosomes (inset 1). Scale bar: 10 μm, scale bar of insets 1 μm (n = 14 cells). SIM images showing the localization of GFP-WDFY2 in relation to mCherry-Rab5 (n = 12 cells), mCherry-Rab4 (n = 13 cells), Anti-Rab7 (n = 14 cells) or mCherry-Rab11 (n = 14 cells). Scale bar: 1 μm, scale bar of insets: 0.5 μm. Protein-Lipid overlay assay using purified full length WDFY2. WDFY2 binds with high selectivity to PtdIns(3)P. Deconvolved widefield image showing GFP-WDFY2 localization to endosomes. GFP-WDFY2-R315A, a mutation in the binding site for PtdIns(3)P, abolishes the localization to endosomes and the protein is cytosolic. Scale bar: 10 μm (n=10 cells).

Article Snippet: The following antibodies were used: Human anti EEA1 provided by Ban-Hock Toh (Monash University), Rabbit anti APPL1 D83H4 from cell signaling (3858S), Rabbit anti Rab7 was from cell signaling (9367), Mouse anti GFP was from Roche (11814 460001), RFP-booster atto 594 was from Chromotek (rba594), Rabbit antibody against Hrs have been described previously , Rabbit anti LAMP1 was from Sigma-Aldrich (L1418), Rhodamine Phalloidin (R415) and Hoechst 33342 (H3570) was from Invitrogen molecular Probes, Goat anti Vps35 (ab10099), Rabbit anti Vps26 (ab23892) were from Abcam.

Techniques: Marker, Protein-lipid Overlay Assay (PLOA), Purification, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: WDFY2 restrains matrix metalloprotease secretion and cell invasion by retention of VAMP3 in endosomal tubules

doi: 10.1101/299610

Figure Lengend Snippet: WDFY2 controls intracellular VAMP3 distribution Deconvolved widefield images of RPE and WDFY2 (-/-) cells transiently expressing GFP-VAMP3 and Cherry-Cortactin. Scale bar: 10 μm (n= 30 cells per condition). Hexagonal superpixel image showing VAMP3 distribution in the cells shown above. Mean intensities of hexagonal ROIs were extracted and the ROI filled with the corresponding mean value, thereby generating superpixels. Distribution of VAMP3 from the nucleus to the leading edge. Cells were transfected with VAMP3 and stained for Cortactin to identify leading edges. A line ROI from the nucleus to the leading edge was drawn, and evenly spaced ROIs were automatically generated. Mean intensity of VAMP3 in each ROI was extracted, normalized for each cell, and then plotted, (n=30 cells for each condition) shown are mean and 95% CI. TIRF micrograph of RPE1 (WT) and WDFY2 (-/-) cells transfected with VAMP3-pHluorin. Individual secretion events (summed over a two minute interval) are indicated with a red circle. Scale bar: 10 μm (n=30 cells per condition). Quantification of VAMP3-pHluorin secretion in RPE1 (WT) and WDFY2 (-/-) cells. Shown are events from 3 experiments (10 cells per experiment per condition). Secretion events per minute are shown normalized to cell area. Student’s unpaired t-test, n=30, p=0.0000029. Quantification of GFP-VAMP3 intensity in EEA1-positive endosomes in RPE1 (WT) and WDFY2 (-/-) cells. Plotted is the mean intensity within EEA1-positive endosomes per cell from 4 experiments (10,12,15,10 cells for (RPE1 (WT)), 10,10,15,10 cells for (WDFY2 (-/-)). Student’s unpaired t-test, n=47/45, p= 0.00035. Quantification of GFP-VAMP3 intensity in Lamp1-positive endosomes in RPE1 (WT) and WDFY2 (-/-) cells. Plotted is the mean intensity within Lamp1-positive vesicles per cell from 4 experiments (10,12,15,10 cells for (RPE1 (WT), 10,10,15,10 cells for (WDFY2 (-/-)). Student’s unpaired t-test, n=47/45, p= 0.00026.

Article Snippet: The following antibodies were used: Human anti EEA1 provided by Ban-Hock Toh (Monash University), Rabbit anti APPL1 D83H4 from cell signaling (3858S), Rabbit anti Rab7 was from cell signaling (9367), Mouse anti GFP was from Roche (11814 460001), RFP-booster atto 594 was from Chromotek (rba594), Rabbit antibody against Hrs have been described previously , Rabbit anti LAMP1 was from Sigma-Aldrich (L1418), Rhodamine Phalloidin (R415) and Hoechst 33342 (H3570) was from Invitrogen molecular Probes, Goat anti Vps35 (ab10099), Rabbit anti Vps26 (ab23892) were from Abcam.

Techniques: Expressing, Transfection, Staining, Generated

Journal: Antioxidants & Redox Signaling

Article Title: H 2 O 2 -Activated Mitochondrial Phospholipase iPLA 2 γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein–Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells

doi: 10.1089/ars.2014.6195

Figure Lengend Snippet: Redox-dependent free FA release in proteoliposomes with recombinant iPLA 2 γ (A–E) and in INS-1E cells (F, G). (A, B, D, E) Fluorimetrically indicated free FA release: Passive diffusion (flip) of FAs into the liposome interior was monitored by sulfopropylquinolinium fluorescence, which increases due to intraliposomal acidification concomitant to FA release. The fluorescence increase was induced by 25 μ M H 2 O 2 (“ +H 2 O 2 ”) in (A, C, D) and was absent without H 2 O 2 ( “no H 2 O 2 ” in A) . Inhibition by 1 μ M r -BEL ( “ +H 2 O 2 +RBEL” in A ), 10 m M glutathione ( “GSH” in D, E), or 10 m M dithiothreitol ( “DTT” in D, E) is also shown. (B) Without iPLA 2 γ (“no iPLA 2 γ”), H 2 O 2 caused no intraliposomal acidification (“ +H 2 O 2 ”) and the response was identical to the baseline (“no H 2 O 2 ”) in contrast to two additions of 10 μ M linoleic acid (“LA”), causing the instant intraliposomal acidification. Five micromolar bovine serum albumin (“BSA”), removing FAs, led to subsequent alkalization back toward the initial pH values. Proteoliposomes contained 0.04 μg of purchased (A, C) or 0.4 μg of the affinity-purified (D, E) recombinant human iPLA 2 γ per mg of phospholipids. (E ) The H 2 O 2 dose response for redox-stimulated FA-induced acidification rate is shown, yielding AC 50 of 0.2 μ M H 2 O 2 , corresponding to 1.4 nmol H 2 O 2 per nmol iPLA 2 γ. (C, F, G) GC/MS quantification of free FAs cleaved during 60 min at 30°C as induced by 25 μ M H 2 O 2 (“H 2 O 2 ”) in iPLA 2 γ–proteoliposomes (C) with 0.04 μg of the purchased protein (Novus); or free FAs cleaved during 2 min at 37°C in INS-1E cells, ntg or silenced as ascribed (F, G) , supplied with 25 m M glucose, as induced by 250 μ M TBHP (F) or 75 nmol PA·l0 −6 cells, corresponding to 0.2 pmol free PA (G) . Indicated FAs were detected in the absence of H 2 O 2 or TBHP (“no add.”), the presence of 25 μ M H 2 O 2 (“H 2 O 2 ”), 250 μ M TBHP (“TBHP”), or 150 μ M PA with 5% BSA; or additional 1 μ M (C) or 40 μ M (F, G) r -BEL (“ + r -BEL”). Averages±s.d. of 3–7 (G) or 4–17 estimations are shown, recalculated relatively to the estimated total content of FA side chains (C) or the initial FA content (F, G) . ANOVA: * p <0.1; ** p <0.05; *** p <0.001. FA, fatty acid; GC/MS, gas chromatography/mass spectrometry; PA, palmitic acid.

Article Snippet: Aliquots in μg·(mg lipid) −1 (0.04 Novus protein; 0.4–0.8 affinity-purified protein) were mixed with phospholipids (Escherichia coli total lipid extract supplemented with 20% bovine heart cardiolipin; Avanti Polar Lipids, Alabaster, AL), to yield 28% cardiolipin, 46% phosphatidylethanolamine, 12% phosphatidylglycerol plus 14% other lipid, and 1 mg·(mg lipid) −1 n -octylpentaoxyethyelene.

Techniques: Recombinant, Diffusion-based Assay, Fluorescence, Inhibition, Affinity Purification, Gas Chromatography-Mass Spectrometry, Gas Chromatography, Mass Spectrometry

Journal: Antioxidants & Redox Signaling

Article Title: H 2 O 2 -Activated Mitochondrial Phospholipase iPLA 2 γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein–Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells

doi: 10.1089/ars.2014.6195

Figure Lengend Snippet: Involvement of mitochondrial iPLA 2 γ in antioxidant protection and amplification of FA-induced insulin release. (A) Antioxidant mechanism based on UCP2 and iPLA 2 γ synergy that is also protective against lipotoxicity in pancreatic β-cells. Acute lipotoxicity leads to FA β-oxidation in mitochondria. Not only the higher substrate load for the respiratory chain (Complexes I, III, IV) contributes to higher superoxide release, but also the electron-transferring flavoprotein:ubiquinone oxido-reductase produces superoxide on β - oxidation . Superoxide can be transformed to H 2 O 2 by superoxide dismutases, SOD2/MnSOD in the matrix, or SOD1/CuZnSOD in the mitochondrial intermembrane space and in the cytosol. Subsequently, H 2 O 2 activates iPLA 2 γ, which cleaves both saturated and unsaturated FAs off IMM phospholipids. Only these nascent FAs induce UCP2-mediated mild uncoupling, which inherently suppresses respiratory chain superoxide formation. Thus, iPLA 2 γ can be also activated by external oxidative stress as well as by redox signaling pathways, elevating H 2 O 2 levels in its vicinity. Ca L , L-type Ca 2+ channel; K ATP , ATP–sensitive K + channel; O 2 •− , superoxide; Rcarn, acylcarnitine; RCoA, acyl-CoA. (B) Involvement of iPLA 2 γ in amplification of FA-induced GPR40 pathway of insulin secretion. After activation of iPLA 2 γ by H 2 O 2 generated due to β-oxidation of incoming palmitic acid, FAs released from mitochondrial membranes provide feedback amplification of GPR40 response, leading to insulin surplus secretion. Lysophospholipids may also be cleaved by specific mitochondrial phospholipase D (mtPLD) . Phosphatidic acid (PhosA) released from normal phospholipids by mtPLD might be further processed by phosphatidic acid preferring PLA1 (PAPLA1). All mentioned lipid metabolites are involved in the glycerol lipid/FA cycle signaling stimulating insulin secretion .

Article Snippet: Aliquots in μg·(mg lipid) −1 (0.04 Novus protein; 0.4–0.8 affinity-purified protein) were mixed with phospholipids (Escherichia coli total lipid extract supplemented with 20% bovine heart cardiolipin; Avanti Polar Lipids, Alabaster, AL), to yield 28% cardiolipin, 46% phosphatidylethanolamine, 12% phosphatidylglycerol plus 14% other lipid, and 1 mg·(mg lipid) −1 n -octylpentaoxyethyelene.

Techniques: Amplification, Transferring, Transformation Assay, Protein-Protein interactions, Activation Assay, Generated