Peroxisomal core structures segregate diverse metabolic pathways

Abstract

Peroxisomes are single membrane-bounded oxidative organelles with various metabolic functions including β-oxidation of fatty acids. Peroxisomes of many species confine certain metabolic enzymes into sub-compartments sometimes visible as electron dense cores. Why these structures form is largely unknown. Here, we report that in the smut fungus Ustilago maydis detergent resistant core structures are enriched for different enzymes excluding several key enzymes of the β-oxidation pathway. This confinement contributes to generation of peroxisome subpopulations that differ in their enzyme content. We identify short amino acid motifs necessary and sufficient for protein self-assembly into aggregates in vitro. The motifs trigger enrichment in cores in vivo and are active in mammalian cells. Perturbation of core assembly via variation of such motifs affects peroxisome function in U. maydis strains challenged with fatty acids. Thus, protein core structures serve to compartmentalize the lumen of peroxisomes thereby preventing interference of biochemical reactions. Metabolic compartmentalization of peroxisomes via assembly of specific proteins may occur in other organisms as well.

Fig. 1. Acyltransferases Mac1 and Mac3 enrich in peroxisome subpopulations and peroxisomal subdomains.

U. maydis strains expressing N-terminally GFP-tagged versions of Mac1 and Mac3 (cyan) and the peroxisomal marker protein mCherry-SKL (magenta) were inspected by epifluorescence microscopy. Representative images of cells grown in glucose (a) or oleate (b). Full images are shown as overlays of two channels. For insets single channels and merged channels are provided. Scale bars: 5 µm. c Quantifications show Pearson’s correlation coefficients of GFP and mCherry signals for indicated strains. Each dot represents one biological repilcate. Center line, mean; error bars, standard error of the mean. Statistical tests were performed by GraphPad Prism via a 1-way Anova combined with a Tukeys post-test to assess significance of the differences. * refers to a p value of 0.0403; ns, not significant p value: 0.2013. Source data are provided as a Source data file. d Cells incubated in medium lacking nitrogen to induce glycolipid biosynthesis were imaged 4 h after inoculation. GFP-Mac1 was expressed under control of its endogenous promoter and the fluorescence signal is depicted in cyan. For insets single channels and merged channels are shown. mCherry-SKL, magenta. Scale bars: 5 µm. e Cells were analyzed by super-resolution microscopy using SIM. Representative images of cells expressing GFP-Mac1 (cyan) and mCherry-SKL (magenta) (left panel), GFP-Mac3 and mCherry-SKL (middle panel), and GFP-SKL and mCherry-SKL (right panel). Full images are shown as overlays of two channels. For insets single channels and merged channels are depicted. Scale bars: 5 µm. f 3D-reconstruction (x,y) of GFP-Mac3 and GFP-SKL (cyan) containing peroxisomes, mCherry-SKL (magenta). Scale bars: 0.5 µm.

Fig. 2. A short peptide motif enhances self-assembly to drive focal enrichment after import.

a Scheme of the transcriptional pulse-chase experiment. Cells were analyzed at indicated time points by super-resolution microscopy using SIM. GFP-Mac3 was compared to GFP-Aox1 (cyan) in strains containing mCherry-SKL. Scale bar: 0.5 µm. b Quantification of colocalization data derived from epifluorescence microscopy depicted in Fig. S3b based on three independent biological replicates. Samples were analyzed directly after (t0), 3 h after (t3), and 6 h after glucose addition (t6). Quantifications show Pearson’s correlation coefficients of GFP and mCherry signals for indicated strains. Center line, median. Significance assessed by an unpaired, two-sided Student’s t-test. **** refer to a p value lower than or equal to 0.0001. c Western blot analysis following the stability of GFP-Mac3 and GFP-Aox1 in a time course experiment. d C-terminal 12 amino acids containing the PTS1 motifs. Cells expressing GFP tagged variants were analyzed by SIM. Representative images of cells are shown. Full images are shown as overlays of two channels. For insets single channels and merged channels are shown. Scale bars: 5 µm. e GFP fused to Thr-Ile-Ile-Val (TIIV) and the PTS1 Ser-Lys-Leu (SKL) was co-expressed with mCherry-SKL. Scale bars: 5 µm. f Cells expressing PTS2-GFP reporter proteins either with or without a C-terminal TIIV motif (cyan) were co-expressed with mCherry-SKL. Quantification showing Pearson’s correlation coefficients of GFP and mCherry signals in Fig. S5e. Scale bars: 5 µm. g Mac3-V₅₇₀R was tagged with GFP (cyan) and localization was analyzed by epifluorescence microscopy. Quantifications in Fig. S6d. Scale bars: 5 µm. h The TIIV motif of Mac3 is embedded in an aggregation prone region. Aggregation of 6xHis-tagged (i) versions of Mac3 and (j) versions of GFP-TIIV and GFP-TIIR was measured over time. Plotted are the means of three replicates for each protein. Different colors denote single experiments. Center line, mean; error bars; standard error of the mean. Significance assessed with an unpaired, two-sided Student’s t-test. *** refer to a p value of 0.0003 (i) and (j). Source data underlying b, c, i, j are provided as a Source data file. GFP, cyan; mCherry, magenta.

Fig. 3. Mac1 and Mac3 self-assemble in detergent resistant cores also containing urate oxidase Uox1.

a Representative epifluorescence images of cells expressing GFP-Uox1 (cyan) and mCherry-SKL (magenta). Full images are shown as overlays of two channels. For insets single channels and merged channels are depicted. Scale bars: 5 µm. b GFP-Uox1 and mCherry-Mac3; organization of figure as described above. Scale bars: 5 µm. c Crude organelle preparations of indicated strains were analyzed by epifluorescence microscopy after incubation in lysis buffer (left) or lysis buffer supplemented with Triton X-100 (right) for 45 min. Organization of pictures as described above. Scale bars: 5 µm. d Transmission electron micrograph of cells expressing GFP-Mac3. Staining was achieved through labeling of GFP-Mac3 with anti-GFP and subsequent immunogold labeling followed by silver enhancement. Scale bar: 0.2 µm. e The experiment was performed as in (c) for the indicated reporter proteins. Scale bars: 5 µm.

Fig. 4. Core assembly enables metabolic compartmentalization of peroxisomes.

a MELs produced from indicated strains were analyzed by TLC. Different MEL variants, which differ in their acetyl-moieties are specified MEL-A—MEL-C. Ustilagic acids (UAs) are a different class of glycolipids produced by U. maydis but not synthesized in peroxisomes. Mac2 is another acyltransferase involved in MEL production, which has overlapping functions with Mac3 and was deleted to facilitate analysis. b Heat maps derived from LC-MS analysis of MELs produced by indicated strains. c Representative epifluorescence images of cells expressing GFP-fused to two additional paralogs of acyl-CoA oxidases (cyan) and mCherry-SKL (magenta). Full images are shown as overlays of two channels. For insets single channels and merged channels are depicted. Scale bar: 5 µm. d Serial dilutions of indicated strains were spotted on minimal media containing either glucose or oleic acid as carbon source. e Magnifications of colonies grown on oleic acid medium show aberrant colony morphology upon expression of the mutated version. Scale bars: 5 µm. f Immunoblot showing expression levels of GFP-Mac3 and GFP-Mac3-V₅₇₀R. Uncropped blots are provided in the Source data file. g Oxygen consumption of cells shifted from glucose to oleic acid. Oxygen concentration was recorded over time and rates were calculated as the first derivative (left). Based on the linear regression of the increase in rates, the oxygen consumption acceleration was calculated (right). Error bars on the data points denote the regression error, while the error bars on the column denote standard deviation of the mean. Significance was assessed with an unpaired, two-sided Student’s t-test. Each line in the graph represents one biological replicate. Source data are provided in the Source data file.

Fig. 5. Diversity and function of peroxisomal cores.

a Representative epifluorescence images of cells expressing GFP-Uox1 or GFP-Uox1-V₁₂₄R and mCherry-SKL. Full images are shown as overlays of two channels. For insets single channels and merged channels are depicted (left). Scale bars: 5 µm. Quantifications show Pearson’s correlation coefficients of GFP and mCherry signals (right). Each dot represents one biological replicate. Center line, mean; error bars, standard error of the mean. Significance was assessed with an unpaired, two-sided Student’s t-test. **** refer to a p value lower than 0.0001. b Aggregation of 6xHis-tagged versions of Uox1 was measured over time. Plotted are the means of three replicates. Quantifications show OD₃₂₀ after 60 min of aggregation. Each dot represents one biological replicate. Center line, mean; error bars, standard error of the mean. Significance was assessed with an unpaired, two-sided Student’s t-test. *** refer to a p value of 0.0002 (right). c Serial dilutions were spotted on minimal media containing glucose or oleic acid. d Magnifications of colonies grown on oleic acid medium show aberrant colony morphology upon expression of the mutated version. Scale bars: 500 µm. e Immunoblot showing expression levels of Uox1 variants. f Activity assay for Uox1 variants. g Oxygen consumption of cells shifted from glucose to oleic acid. Based on the linear increase in consumption rates (left), the oxygen consumption acceleration rate was plotted (right). Error bars on data points denote the regression error, while the error bars on the column denote standard deviation of the mean. Significance was assessed with an unpaired, two-sided Student’s t-test. h Quantification shows the mean value of Pearson’s correlation coefficients of GFP signals and mCherry signals. Quantifications for three biological replicates for each of the analyzed candidate proteins are shown in Fig. S10a–c. i Two candidate proteins—D-amino oxidase 1 (Dao1) and malate synthase 1 (Mls1)—were co-expressed with mCherry-SKL. Representative epifluorescence images are shown. Full images are provided as overlays. For insets single channels and merged channels are depicted. Scale bars: 5 µm. Source data underlying a, b, e, g, h, i are provided in the Source data file. GFP, cyan; mCherry, magenta.

Fig. 6. Conservation of core formation properties.

a Mus musculus urate oxidase (MmUOX1) was tagged with GFP (cyan) and expressed in U. maydis cells either co-expressing mCherry-SKL (magenta) or mCherry-Mac3 (magenta). b Crude organelle preparations of indicated strains were imaged by epifluorescence microscopy after incubation in lysis buffer supplemented with Triton X-100 (right). Preparations incubated in lysis buffer without Triton X-100 served as control (left). c Constructs expressing GFP-TIIV-SKL and mCherry-SKL were transfected into RPE1 cells. Cells were analyzed by SIM. The representative picture shows an overlay of the GFP-signal (cyan) and the mCherry-signal (magenta) in the overview. The signal of single channels as well as the overlay are depicted for magnified insets. d M. musculus urate oxidase (MmUox1) was tagged with mCherry (magenta) and co-expressed with GFP-TIIV-SKL (cyan) in RPE1 cells. Cells were analyzed by SIM. Representative pictures are organized as above. Scale bars: 5 µm.

Fig. 7. Working model—peroxisome cores may serve for metabolic compartmentalization.

Uox1 and Mac3 assemble in detergent resistant cores, while several β-oxidation enzymes are distributed homogeneously in the entire peroxisomal lumen (left). Variation of motifs for self-assembly affected localization of Uox1 and Mac3 and growth of cells challenged with oleic acid medium (right).