Peroxisomes form intralumenal vesicles with roles in fatty acid catabolism and protein compartmentalization in Arabidopsis

Abstract

Peroxisomes are vital organelles that compartmentalize critical metabolic reactions, such as the breakdown of fats, in eukaryotic cells. Although peroxisomes typically are considered to consist of a single membrane enclosing a protein lumen, more complex peroxisomal membrane structure has occasionally been observed in yeast, mammals, and plants. However, technical challenges have limited the recognition and understanding of this complexity. Here we exploit the unusually large size of Arabidopsis peroxisomes to demonstrate that peroxisomes have extensive internal membranes. These internal vesicles accumulate over time, use ESCRT (endosomal sorting complexes required for transport) machinery for formation, and appear to derive from the outer peroxisomal membrane. Moreover, these vesicles can harbor distinct proteins and do not form normally when fatty acid β-oxidation, a core function of peroxisomes, is impaired. Our findings suggest a mechanism for lipid mobilization that circumvents challenges in processing insoluble metabolites. This revision of the classical view of peroxisomes as single-membrane organelles has implications for all aspects of peroxisome biogenesis and function and may help address fundamental questions in peroxisome evolution.

Fig. 1. Membrane reporters reveal inner peroxisomal membranes that are not endosomes or autophagosomes.

a, b Z-projections of peroxisomes imaged using mRuby3 channel (magenta) of 8-day-old seedling cotyledon epidermal cells expressing mNeonGreen-mPTS^PEX26 (a) or mPTS^PEX22-mNeonGreen (b) and mRuby3-PTS1. c Merged mNeonGreen-mPTS^PEX26 (green) and mRuby3-PTS1 (magenta) channels from (a). d Cross-section of peroxisome boxed in (c). e Merged mPTS^PEX22-mNeonGreen (green) and mRuby3-PTS1 (magenta) channels from (b) reveals peroxisomes and fluorescence resembling ER. f Cross-section of peroxisome boxed in (e). g Z-projection of 4-day-old cotyledon epidermal cells expressing mNeonGreen-mPTS^PEX26 (green) and mRuby3-PTS1 (magenta) show ILVs in all peroxisomes with a resolvable lumen. h Cross-section of peroxisome boxed in (g). i Z-projection of 3-day-old cotyledon epidermal cells expressing mPTS^PEX22-mNeonGreen (green) and mRuby3-PTS1 (magenta) shows ILVs in peroxisomes and extra-peroxisomal fluorescence resembling ER. j Cross-section of peroxisome boxed in (i). k Cross-section of cotyledon cell expressing mPTS^PEX26-mNeonGreen (green) and mRuby3-PTS1 (magenta) showing inner peroxisomal membranes with tubular morphology. l Cross-section of cotyledon epidermal cells expressing mNeonGreen-mPTS^PEX26 (green) and stained with FM4-64 (magenta), which marks plasma membrane and endosomes (e.g., arrowheads). m Z-projection of cotyledon epidermal cells of atg7-4 expressing mNeonGreen-mPTS^PEX26 (green) and mRuby3-PTS1 (magenta) showing abundant peroxisomal ILVs remain when autophagy is prevented. n Cross-section of hypocotyl cells expressing mRuby3-PTS1 (magenta) without a membrane reporter showing nonfluorescent “holes” in peroxisomes.

Fig. 2. Peroxisome size decreases as inner membrane density increases.

a, b Z-projections of cotyledon epidermal cells of a 4-day-old seedling expressing mNeonGreen-mPTS^PEX26 (green) and mRuby3-PTS1 (magenta) imaged with high-magnification spinning disk confocal microscopy over 5 h (a) or lower-magnification scanning confocal microscopy over 9 h (b). Individual peroxisomes are designated with differently colored arrowheads. c Diameters of individual peroxisomes marked with arrowheads of same color in panel (b).

Fig. 3. ESCRT function is required for efficient peroxisomal inner membrane formation.

a Diagram of dominant-negative ESCRT constructs driven by the β-estradiol-inducible pXVE promoter system. b Dominant-negative ESCRT derivatives exhibit estradiol-dependent accumulation and do not impair peroxisomal protein processing. Immunoblots of 5-day-old seedlings grown without or with 25 µM β-estradiol are shown. The PTS2 signal region of the PMDH precursor is cleaved from mature PMDH in the peroxisome lumen. Precursor accumulation in the pex12-1 mutant reflects protein import defects. c ESCRT disruption impairs peroxisomal ILV formation. Cotyledon epidermal cells are shown of 5-day-old seedlings expressing mNeonGreen-mPTS^PEX26 (green) and mRuby3-PTS1 (magenta) and lacking (control) or carrying constructs expressing dominant-negative SNF7^L22W or VPS4^E232Q grown with DMSO (mock) or β-estradiol. d ESCRT disruption causes enlarged peroxisomes. Peroxisome diameter was quantified from images of four seedlings (Supplementary Fig. 2) expressing mNeonGreen-mPTS^PEX26 and mRuby3-PTS1 and grown without (gray) or with (blue) 25 µM β-estradiol to induce SNF7^L22W or VPS4^E232Q expression. Boxes show second and third quartiles, horizontal lines are medians, and whiskers encompass data points up to 1.5 times the interquartile range past the box ends. Student’s two-tailed unpaired t tests were used to compare peroxisome size with and without β-estradiol.

Fig. 4. Efficient peroxisomal ILV formation requires β-oxidation.

a, b Enlarged peroxisomes in mutants with impaired β-oxidation show disrupted ILV formation. Wild-type seedlings and various mutants with impaired β-oxidation expressing mNeonGreen-mPTS^PEX26 (green) and mRuby3-PTS1 (magenta) were imaged over several days; cross-sections (a) and peroxisome diameters (b) are shown. Boxes show second and third quartiles, horizontal lines are medians, and whiskers encompass data points up to 1.5 times the interquartile range past the box ends. c Magnified peroxisome cross-sections show ILVs in wild-type peroxisomes dispersed in the lumen and residual ILVs in pxn-4, acx2-2, and mfp2-8 aggregated near the outer membrane. d Z-projections of peroxisomes in root cells show wild-type peroxisomes with densely packed inner membranes and pxa1-1 and pxn-4 pxa1-1 peroxisomes lacking most inner membranes.

Fig. 5. Peroxisomal vesicles lack engulfed lipid droplets.

a Dominant-negative ESCRT induction impairs lipid mobilization. Z-projections of cotyledon epidermal cells of seedlings expressing mNeonGreen-mPTS^PEX26 (green) and mRuby3-PTS1 stained with monodansylpentane (MDH) to mark lipid droplets (magenta) and grown with DMSO (mock) or β-estradiol to induce SNF7^L22W expression. b Lipid droplet diameter in SNF7^L22W seedlings from panel (a). Boxes show second and third quartiles, horizontal lines are medians, and whiskers encompass data points up to 1.5 times the interquartile range past the box ends. Student’s two-tailed unpaired t tests were used to compare lipid droplet size with and without β-estradiol. c Peroxisomal ILVs do not accumulate neutral lipids. Cross-section cotyledon epidermal cells from wild-type or acx2-2 expressing mNeonGreen-mPTS^PEX26 (green) and lipid droplets stained with MDH (magenta).

Fig. 6. Peroxisomal vesicles compartmentalize proteins.

a Cross-section of 4-day-old cotyledon epidermal cell expressing mNeonGreen-mPTS^PEX26 (green) and mRuby3-PTS1 (magenta) shows peroxisomal ILVs enclosing (yellow arrowhead) or lacking (blue arrowheads) PTS1 reporter. b, c Cross-sections of 4-day-old cotyledon epidermal cells expressing mRuby3-PTS1 (magenta), mTagBFP2-SCO3 (blue), and mNeonGreen-mPTS^PEX22 (green) (b) or mPTS^PEX26-mNeonGreen (green) (c) show SCO3 accumulation (arrowheads) in a subset of ILVs. d, e Cross-sections of 4-day-old cotyledon epidermal cells expressing mRuby3-PTS1 (magenta), mTagBFP2-UP9 (blue), and mNeonGreen-mPTS^PEX22 (green) (d) or mPTS^PEX26-mNeonGreen (green) (e) show UP9 accumulation (arrowheads) in a subset of ILVs. f Cross-section of 3-day-old cotyledon epidermal cells expressing mNeonGreen-mPTS^PEX26 and mRuby3-PTS1 (magenta) shows ILVs with (green) and without (arrowheads) mNeonGreen-mPTS^PEX26 membrane fluorescence.