Journal: Autophagy

Article Title: The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders

doi: 10.1080/15548627.2017.1291470

Figure Lengend Snippet: PEX5 accumulates on the peroxisomal membrane in a ubiquitinated form and signals for pexophagy in cells depleted of peroxisomal AAA-complex components. (A) Immunoblots of the total cell lysates for PEX5 from HeLa cells transfected with plasmids encoding GFP-ubiquitin (GFP-Ub) and RFP-SKL after 2 d of treatment with siRNA as indicated. Cells were also treated with 10 μm chloroquine for 24 h to prevent peroxisome loss. The bracket represents modified PEX5; the arrow indicates PEX5, whereas the asterisks (*, **) indicate nonspecific bands. (B) Graph of the relative densitometry reading of GFP-Ub x -PEX5:PEX5 ratio; where GFP-Ub x -PEX5 represent the higher molecular weight bands indicated in (A). The average (n = 3) ± standard deviation for each condition is shown. Asterisks represent p-values of statistics relative to siCTRL: *p < 0.05. A.U., arbitrary units. (C) Same as (A) but immunoblot for ABCD3. The top dashed arrow represents modified ABCD3; the bottom solid arrow indicates ABCD3. (D) Representative fluorescence images of HeLa cells transfected with siRNA as indicated over 2 consecutive d and immunostained for ABCD3 and PEX5. Scale bars: 20 μm. (E) Graph of the average density of PEX5 punctate structures within a cell (the number of PEX5 puncta per volume of cell [μm ]) of 30 cells per trial (n = 3) ± standard deviation in (D). Asterisks represent p values compared with siCTRL: *p < 0.05. (F) Subcellular fractionation of HeLa cells transfected with GFP-ubiquitin (GFP-Ub) and RFP-SKL plasmids after 2 d of treatment with siRNA and chloroquine as in (A). The post nuclear homozygous lysates (WCL) were fractionated into cytosol and membrane fractions, and immunoblotted with the indicated antibodies. The top dashed arrow represents modified PEX5; the bottom solid arrow indicates PEX5, whereas the asterisk (*) indicates nonspecific bands. (G) Representative fluorescence images of HeLa cells transfected with nontargeting siRNA (siCTRL), siRNA against PEX1 (si PEX1 ), and PEX26 (si PEX26 ), with or without siRNA against PEX5 (si PEX5 ) as indicated and immunostained for ABCD3. Scale bars: 50 μm. (H) Graph of the average ABCD3 density per cell of at least 30 cells per trial (n = 3) ± standard deviation in (G). Asterisks represent p-values: *p < 0.05, **p < 0.01.

Article Snippet: The rabbit polyclonal anti-PEX5 antibody used for immunoblotting (1:1000) was generated by immunizing the New Zealand white rabbit with full-length PEX5–6xHIS (accession #NM_001131023) by standard protocol (Cedarlane, Burlington, Ontario).

Techniques: Membrane, Western Blot, Transfection, Ubiquitin Proteomics, Modification, Molecular Weight, Standard Deviation, Fluorescence, Fractionation

Journal: Autophagy

Article Title: The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders

doi: 10.1080/15548627.2017.1291470

Figure Lengend Snippet: A model for AAA-dependent pexophagy. The peroxisomal AAA-complex prevents ubiquitin-dependent pexophagy and enables peroxisomal matrix protein import. (A) Cells under basal conditions possess their peroxisomal AAA-complex, consisting of PEX1, PEX6 and PEX26, allowing for the efficient removal of ubiquitinated PEX5. Import of matrix proteins is represented by -PTS1. (B) Defect in any of the AAA-complex genes results in the loss of AAA-complex function and the accumulation of ubiquitinated PEX5. This results in the recruitment of autophagy receptors, NBR1 and SQSTM1, which then results in targeting of peroxisomes to phagophores for degradation via pexophagy. (C) Inhibiting pexophagy with chloroquine (CQ) prevents the degradation of peroxisomes, increasing their half-life, and allowing for the import of matrix proteins. This increase in peroxisome numbers and their subsequent increase in import of peroxisomal matrix enzymes results in improved peroxisomal function.

Article Snippet: The rabbit polyclonal anti-PEX5 antibody used for immunoblotting (1:1000) was generated by immunizing the New Zealand white rabbit with full-length PEX5–6xHIS (accession #NM_001131023) by standard protocol (Cedarlane, Burlington, Ontario).

Techniques: Ubiquitin Proteomics