ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

Abstract

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO-ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A-binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5-VAPB interaction regulates PO-ER associations. Moreover, we demonstrate that loss of PO-ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO-ER associations in mammalian cells and report a new function for ACBD5 in PO-ER tethering.

Figure 1.

ACBD5 interacts with VAPB. (A) Schematic overview of VAPB and ACBD5 domain structure. Mutations in acyl-CoA binding and FFAT-like motifs are indicated. (B) Immunoprecipitation (IP) of GFP-ACBD5 and Myc-VAPB after coexpression in COS-7 cells. GFP was used as control. Samples were immunoprecipitated (GFP-Trap) and immunoblotted using Myc-GFP antibodies. (C) In vitro binding assay using GST-VAPB and MBP fusions of ACBD5 (AcB, mutations in the acyl-CoA binding motif; FFAT, mutations in FFAT motif) expressed in E. coli. GST served as control. Samples were immunoprecipitated using GST-Trap and immunoblotted using MBP-GST antibodies. (D) Model of ACBD5–VAPB interaction.

Figure 2.

ACBD5–VAPB coexpression increases PO–ER association. (A–F) COS-7 cells were transfected with Myc-VAPB alone (A) or Myc-VAPB coexpressed with GFP-ACBD5, FLAG-ACBD5, FLAG-ACBD5-AcB, FLAG-ACBD5-FFAT, and GFP-Sec61β (B–F). Myc-VAPB is labeled in red and Pex14/ACBD5 in green, except in F, where ACBD5 is in red (B–D, arrows highlight PO–ER association; A, E, and F, arrows highlight lack of PO–ER association). (G) Quantification of overlap of PO–ER fluorescent signals. Note this analysis has limitations because of the proximity of POs and the ER. αPEX14, PO marker. Data are presented as mean ± SEM. Bars: (main) 20 µm; (insets) 5 µm.

Figure 3.

ACBD5 and VAPB mediate PO–ER association. (A) Representative electron micrographs of PO–ER associations in COS-7 cells transfected with control vector, ACBD5, VAPB, and ACBD5 + VAPB (arrowheads highlight close ER–PO association). Electron-dense material between the PO–ER membranes is visible (see arrows in enlargement). (B) Quantitative analysis of the mean fraction of POs associated with the ER. (C) Assessment of the mean PO membrane surface in direct contact with the ER membrane. (D) Representative electron micrographs of PO–ER associations in HepG2 cells treated with control, ACBD5, VAPB, and ACBD5 + VAPB siRNAs. Arrowheads in overview mark POs with limited or no ER contact. (E) Quantitative analysis of mean fraction of POs associated with the ER after siRNA treatments. (F) Assessment of mean PO–ER membrane contact after siRNA treatment. (G) Immunoblot showing ACBD5 and VAPB signals after silencing with correspondent siRNAs. GAPDH, loading control. Data were analyzed by one-way analysis of variance with Tukey’s multiple comparison test; ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001. Error bars represent SEM, with three to six experiments per condition. Bars: (main) 200 nm; (zoom) 50 nm. M, mitochondrion; N, nucleus.

Figure 4.

ACBD5/VAPB interaction influences PO migration in gradients and PO motility in human fibroblasts. (A) ACBD5/VAPB coexpression alters PO distribution in density gradients. PO-enriched fractions, prepared from HepG2 cells (Control) and cells cotransfected with GFP-rACBD5 and Myc-VAPB, were separated in continuous Nycodenz-gradients. Distribution of organelle markers was assessed by immunoblotting of fractions. Coexpression of ACBD5 and VAPB shifts POs to lower densities, similar to ER markers (compare boxed regions). Pex14 (PO); ACOX1, acyl-CoA oxidase 1 (PO); ATP synt. a, ATP synthase α subunit (MITO); PDI, protein disulfide isomerase (ER). (B–H) Loss of ACBD5 increases PO movement. Human fibroblasts were treated with control (Cont) or ACBD5 siRNA, transfected with GFP-PTS1, and analyzed by live cell imaging (Videos 1 and 2). (B–D) Trajectory plots. 100 PO trajectories were retrieved for each condition and the first 20 time frames plotted starting at a center. (E–G) Density plots. The x and y coordinates of all trajectories ≥20 time frames were pooled and binned in the interval −3,3 µm in x and y directions, using 50 bins. The log-scaled 2D histogram of these points was plotted using “jet” color map. (H) ECDF plots. Instantaneous trajectory speed profiles were estimated by calculating distance moved between each time point in the trajectory. These speeds were pooled and converted to an empirical cumulative distribution function (ECDF). By pooling speeds for all datasets for a given condition, a single ECDF was generated for each (minimum of 38,175 trajectories from 24 videos per condition).

Figure 5.

Loss of ACBD5 or VAPB reduces PO membrane expansion in Mff-deficient fibroblasts. PO morphology in Mff-deficient fibroblasts (control; A) after reintroduction of Mff (B) or silencing of Pex11β (C), ACBD5 (D), or VAPB (E). Fixed cells were labeled with anti-Pex14 antibodies. (F) Quantification of PO morphology in controls and silenced cells (n = 2,500, from three independent experiments). Data are presented as mean ± SEM. ***, P < 0.001; ns, not significant. (G) Immunoblots of cell lysates. Loading controls used were catalase (Cat), GAPDH, and thioredoxin (Thiored). (H–M) Localization of endogenous ACBD5 in Mff-deficient fibroblasts. Fixed cells labeled with anti-ACBD5 and anti-catalase antibodies. Arrowheads denote ACBD5 concentrated at globular POs that give rise to tubular membranes. Bars, 10 µm.