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Peripheral myelin protein 22 alters membrane architecture

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Science Advances  05 Jul 2017:
Vol. 3, no. 7, e1700220
DOI: 10.1126/sciadv.1700220
  • Fig. 1 PMP22 forms ordered assemblies upon reconstitution into lipid vesicles.

    (A to C) Examples of protein-lipid MLAs created when PMP22 is reconstituted into 4:1 POPC/ESM vesicles via the dialysis method and visualized by negative stain EM. (D) Representative image of multilamellar vesicles (MLVs) prepared in the absence of protein via the dialysis method [lipid-only control (LOC)]. (E) MLVs prepared by spontaneous bilayer formation through hydration of lipids with water. (F) Control assemblies containing 4:1 POPC/ESM and the tetraspan VSD of KCNQ1 reconstituted via the dialysis method. Scale bars (all panels), 100 nm. (G) Quantification of the relative percentage of MLAs present in a series of negative stain EM images of wild-type (WT) PMP22, LOC, MLVs, and the tetraspan VSD domain of KCNQ1. All individual object counts were converted to percentage of total counts for a particular sample and were normalized to the percentage of total counts represented by MLAs in the WT PMP22 control, which was set to 1.0. Green, WT; red, LOC; blue, MLV; orange, VSD. (H) Sucrose gradient analysis of PMP22 reconstituted for 10 days without (−; top) or with (+; bottom) lipids. Fractions were collected from top (low density) to bottom (high density) and analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) with silver staining.

  • Fig. 2 Differences between MLVs and MLAs are visible by cryo-EM.

    (A and B) Representative images of vitrified MLVs prepared in the absence of protein via the dialysis method (A) or by spontaneous bilayer formation through hydration of lipids only with water (B). (C and D) Examples of MLAs created when PMP22 is reconstituted into 4:1 POPC/ESM vesicles via the dialysis method and visualized using cryo-EM. Scale bars (all panels), 100 nm.

  • Fig. 3 MLAs examined by cryo-ET.

    (A) Representative tomographic slices (1.47 nm) of two MLAs. *, MLAs in image. (B and C) Two MLAs from (A). Arrowheads indicate the ends of MLA. (D and E) Segmentation view of the corresponding MLA from (B) and (C). Scale bars, 100 nm (A, B, and D) and 50 nm (C and E). (F) Model demonstrating the compressed wrapped membranes of an MLA. (G) Model demonstrating the nesting vesicles of MLVs.

  • Fig. 4 Altered PMP22 LPRs disrupt MLA formation.

    Representative negative stain images of PMP22 reconstitution assays carried out at LPRs (w/w) of 0.5 (A and B), 1.0 (C and D), and 10.0 (E and F). Scale bars (all panels), 100 nm. (G) Quantification of the relative percentage of MLAs present in a series of negative stain EM images of WT PMP22 reconstitutions at LPRs (w/w) of 0.5, 1.0. 2.0, 4.0, and 10.0. All individual object counts were converted to the percentage of total counts for a particular sample and were normalized to the percentage of counts represented by MLAs in the LPR 1.0 sample, which was set to 1.0. Red, LPR 0.5; green, LPR 1.0; blue, LPR 2.0; orange, LPR 4.0; purple, LPR 10.0. Error bars represent SEM between biological replicates. *P < 0.05, **P < 0.01. Statistical significance is only indicated for MLAs.

  • Fig. 5 MLA formation is not dependent on intermolecular disulfide linkage.

    (A and B) Representative negative stain images of MLAs formed in a reconstitution assay using a Cys-less PMP22 mutant (C42S, C53S, C85A, and C109A). Scale bars (A and B), 100 nm. (C) Quantification of the percentage of MLAs present in a series of negative stain EM images of WT and Cys-less PMP22 reconstitutions. All individual object counts were converted to the percentage of total counts for a particular sample and were normalized to the percentage of total counts represented by MLAs in the WT PMP22 control, which was set to 1.0. Green, WT control; red, Cys-less PMP22.

  • Fig. 6 ECL1 and ECL2 are important for MLA formation.

    (A) Quantification of the relative percentage of MLAs present in a series of negative stain EM images of PMP22 reconstitutions of WT PMP22 only, WT PMP22 incubated with GST-ECL1, and WT PMP22 incubated with GST-ECL2. Green, WT control; light blue, GST-ECL1 + WT PMP22 (1:1 molar ratio); dark blue, GST-ECL1 + WT PMP22 (4:1 molar ratio); light orange, GST-ECL2 + WT PMP22 (1:1 molar ratio); dark orange, GST-ECL2 + WT PMP22 (4:1 molar ratio). Error bars represent SEM between biological replicates. **P < 0.01. Statistical significance is only indicated for MLAs. (B) Quantification of the relative percentage of MLAs present in a series of negative stain EM images of PMP22 reconstitutions of WT PMP22; ECL1 loop-mutants PMP22 D37K, L38A, or W39A; and ECL2 loop-mutant PMP22 W124A. Green, WT control; red, D37K; blue, L38A; orange, W39A; purple, W124A. For both panels, all individual object counts were converted to the percentage of total counts for a particular sample and were normalized to the percentage of total counts represented by MLAs in the WT PMP22 control. All values were normalized to the percentage of WT control MLAs, which was set to 1.0.

  • Fig. 7 The L16P PMP22 (TrJ) mutation disrupts MLA formation.

    (A) Quantification of the relative percentage of MLAs present in a series of negative stain EM images in both WT and L16P PMP22 reconstituted on the same day. All individual object counts were converted to the percentage of total counts for a particular sample and were normalized to the percentage of total counts represented by MLAs in the WT PMP22 control, which was set to 1.0. Green, WT control; red, L16P. (B and C) Representative negative stain EM images of the disordered MLAs found in L16P PMP22 reconstitutions. Scale bars (B and C), 100 nm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/7/e1700220/DC1

    movie S1. Cryo-ET of an MLA showing that it is composed of compressed wrapped vesicles (corresponding to Fig. 3B).

    movie S2. Cryo-ET of an MLA showing that it is composed of compressed wrapped vesicles (corresponding to Fig. 3C).

    movie S3. Cryo-ET of an MLV showing that it is composed of nested vesicles rather than compressed wrapped vesicles seen in MLAs (corresponding to fig. S3B).

    fig. S1. Representative examples of objects observed in reconstitution experiments by negative stain.

    fig. S2. Examples of interperiod repeat distance measurements taken for MLAs and MLVs imaged with cryo-EM.

    fig. S3. MLV vesicles examined by cryo-ET.

    fig. S4. Schematic showing the organization of PMP22.

    fig. S5. Addition of GST does not reduce MLA formation.

    table S1. Total counts, percentages, and SDs of WT PMP22 reconstitutions at the LPR of 1.0.

    table S2. Total counts and normalized values from images of PMP22 reconstitutions compared to protein-free and KCNQ1 potassium channel voltage sensor domain (Q1-VSD) controls.

    table S3. Total counts and normalized values from images of WT PMP22 reconstitutions at different LPRs.

    table S4. Total counts and normalized values from images of reconstitutions of WT and Cys-less PMP22.

    table S5. Total counts and normalized values from images of PMP22 reconstitutions containing GST-ECL1 and GST-ECL2.

    table S6. Total counts and normalized values from images of PMP22 reconstitutions containing WT or GST.

    table S7. Total counts and normalized values from images of reconstitutions containing only lipids and GST, GST-ECL1, or GST-ECL2.

    table S8. Total counts and normalized values from images of reconstitutions of WT PMP22 compared to PMP22 constructs with mutations in conserved residues of ECL1 and ECL2.

    table S9. Total counts and normalized values from images of reconstitutions of WT and the TrJ mutant PMP22 construct.

  • Supplementary Materials

    This PDF file includes:

    • Legends for movies S1 to S3
    • fig. S1. Representative examples of objects observed in reconstitution experiments by negative stain.
    • fig. S2. Examples of interperiod repeat distance measurements taken for MLAs and MLVs imaged with cryo-EM.
    • fig. S3. MLV vesicles examined by cryo-ET.
    • fig. S4. Schematic showing the organization of PMP22.
    • fig. S5. Addition of GST does not reduce MLA formation.
    • table S1. Total counts, percentages, and SDs of WT PMP22 reconstitutions at the LPR of 1.0.
    • table S2. Total counts and normalized values from images of PMP22 reconstitutions compared to protein-free and KCNQ1 potassium channel voltage sensor domain (Q1-VSD) controls.
    • table S3. Total counts and normalized values from images of WT PMP22 reconstitutions at different LPRs.
    • table S4. Total counts and normalized values from images of reconstitutions of WT and Cys-less PMP22.
    • table S5. Total counts and normalized values from images of PMP22 reconstitutions containing GST-ECL1 and GST-ECL2.
    • table S6. Total counts and normalized values from images of PMP22 reconstitutions containing WT or GST.
    • table S7. Total counts and normalized values from images of reconstitutions containing only lipids and GST, GST-ECL1, or GST-ECL2.
    • table S8. Total counts and normalized values from images of reconstitutions of WT PMP22 compared to PMP22 constructs with mutations in conserved residues of ECL1 and ECL2.
    • table S9. Total counts and normalized values from images of reconstitutions of WT and the TrJ mutant PMP22 construct.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mov format). Cryo-ET of an MLA showing that it is composed of compressed wrapped vesicles (corresponding to Fig. 3B).
    • movie S2 (.mov format). Cryo-ET of an MLA showing that it is composed of compressed wrapped vesicles (corresponding to Fig. 3C).
    • movie S3 (.mov format). Cryo-ET of an MLV showing that it is composed of nested vesicles rather than compressed wrapped vesicles seen in MLAs (corresponding to fig. S3B).

    Files in this Data Supplement:

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