Overview of the screening of the Danio rerio proteome to identify candidate proteins with a C-terminal peroxisomal targeting sequence (PTS1). See Materials and Methods for details.

Schematic overview of the predicted molecular machineries and D. rerio proteins localized at the membranes of peroxisomes in zebrafish. See text for further details. Matrix protein import: after synthesis on free ribosomes, cargo proteins containing the peroxisomal targeting signals PTS1 or PTS2 bind to the corresponding cytosolic receptors Pex5 (E7FGF7) or Pex7 (A8KBW8) (Pex-proteins are indicated as sole numbers) and form receptor–cargo complexes. The Pex7–cargo complex requires accessory factors for import (e.g., Pex5L, a long isoform of Pex5). Import is achieved by a complex set of integral or peripheral PMPs that form the matrix protein import machinery, which mediates docking of the cargo-bound import receptor at the peroxisomal membrane [Pex13 (Q6PFQ3), Pex14 (A0A2R8QMZ2)], cargo translocation into the matrix of the organelle by a dynamic translocon [Pex2 (E7F4V8), Pex10 (Q5XJ92), Pex12 (B0R157)], and export of the receptor back to the cytosol [Pex1 (A0A0R4IPF0), Pex6 (F1QMB0)]. Recycling of the receptor involves its ubiquitination (Ub) and extraction from the membrane by an AAA–ATPase complex (Pex1, Pex6). Pex6 binds to the membrane protein Pex26 (F1RBL0). Membrane assembly and insertion of PMPs (containing an mPTS) depend on Pex19 (F1R313), Pex3 (Q5RIV3), and Pex16 (F1RDG2). Pex19 functions as a cycling receptor/chaperone, which binds the PMPs in the cytosol and interacts with Pex3 at the peroxisomal membrane. Proliferation, growth and division: Pex11α (A3QJY9), Pex11β (Q0P453), and Pex11γ (Q4V8Z0) are involved in the regulation of peroxisome size and number (proliferation). Pex11β remodels the peroxisomal membrane and interacts with the membrane adaptors Mff (F1Q877; A8E7S0) and Fis1 (A0A2R8Q8G0), which recruit the dynamin 1-like fission GTPase Dnm1l to peroxisomes, which in mammals is activated by Pex11β. Motility: mammalian peroxisomes move along microtubules, and Miro/Rhot (Rhot1a, Q6NVC5; Rhot1b, A0A0R4IGX0; Rhot2, Q32LU1) serves as membrane adaptor for the microtubule-dependent motor proteins kinesin and dynein. Tethering: Acbd5 (E9QCH6; A5WV69) and Acbd4 (F1QA31) interact with ER-resident Vap (Vapb, Q6P2B0) to mediate peroxisome–ER contacts. Metabolite transport: uptake of fatty acids in D. rerio peroxisomes is mediated by ABC transporter proteins (Abcd1, F1RBC8; Abcd2, E7F973; Abcd3a, A0A0R4IRL4; Abcd3b, B0UY91). Other peroxisomal transporter and membrane proteins in zebrafish include (functions are in part unclear): PMP34 (Slc25a17) (A5D6T2), a peroxisomal CoA transporter; PMP52 (Tmem135) (A4QN71) and PMP24 (Pxmp4) (A0A2R8QFW3) belong to the Tim17 family of transporters; PMP22 (Pxmp2) (Q66HU7); Slc27a2/4 (F1QQC5, Q1ECW0; Q567D7), acyl-CoA synthetase long chain family member; Marc (A0A2R8PWS6), mitochondrial amidoxime reducing component; Atad1 (A0A2R8RM20, B2GP29), ATPase family AAA (ATPase associated with various cellular activities) domain-containing protein 1 with a potential role in dislocation/quality control of tail-anchored membrane proteins; Aldh3a2 (A0A2R8PW97, E9QH31), fatty aldehyde dehydrogenase; Far1/2 (A0A0R4ICF6/Q1L8Q4), fatty acyl-CoA reductase 1/2 (ether lipid biosynthesis); Mavs (F1REK4), mitochondrial antiviral signalling protein with a putative role in innate immune response; Trim37 (E7FBZ8), tripartite motif-containing protein 37, an E3 ubiquitin-protein ligase involved in Pex5 mediated peroxisomal matrix protein import; Usp30 (A2BGT0), ubiquitin-specific protease 30, a deubiquitinase involved in the turnover of peroxisomes; Pnpla8 (F1RE62), Patatin-like phospholipase domain-containing 8. Proteins with a potential dual localization to both peroxisomes and mitochondria are marked with an asterisk. Pex, peroxin; PMP, peroxisomal membrane protein (adapted from Islinger et al., 2018, but containing the zebrafish specific nomenclature).

Schematic representation of the pathways of fatty acid β-oxidation (A), ether phospholipid synthesis (B) and purine catabolism (C) in peroxisomes. (A) Peroxisomes degrade fatty acids in four consecutive steps: (1) oxidation, (2) hydration, (3) dehydrogenation, and (4) thiolytic cleavage. Step 1 is performed by multiple acyl-CoA oxidases (1-n) with different substrate specificities. Steps 2 and 3 involve two bifunctional proteins (L-BP, D-BP) harbouring both enoyl-CoA hydratase and 3-hydroxy-acyl-CoA dehydrogenase activity. Step 4 requires different thiolases (e.g., ACAA1, SCPx). Peroxisomes have acquired a set of accessory enzymes (e.g., for fatty acid α-oxidation) to transform the acyl-CoA esters of those fatty acids (FA), which cannot directly enter the β-oxidation pathway (e.g., phytanic acid) (see text for details). (B) The first three steps of ether phospholipid synthesis take place in peroxisomes; synthesis continues in the endoplasmic reticulum (ER). (C) Enzymes involved in purine catabolism in different vertebrate groups and their excretion products. Note that the cellular localisation (cytosolic, peroxisomal) of these enzymes varies amongst vertebrate species. Xanthine oxidase is cytosolic (Cyt) in H. sapiens (Hs) and D. rerio (Dr). Urate oxidase, HIUase, OHCU decarboxylase, and allantoicase are supposed to localise to peroxisomes (PO) in D. rerio, but are absent in H. sapiens (asterisks) (see also Table 1). Allantoinase is cytosolic in D. rerio, but absent in H. sapiens (asterisks). AADHAPR, alkyl-dihydroxyacetone phosphate reductase; ADHAPS, alkyl-dihydroxyacetone phosphate synthase; DHAP, dihydroxyacetone phosphate; DHAPAT, dihydroxyacetone phosphate acyltransferase; FAR1/2, fatty acyl-CoA reductases 1/2; HIUase, 5-hydroxyisourate hydrolase; OHCU decarboxylase, 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase (adapted from Islinger et al., 2010).

Comparison of PTS1 motifs between D. rerio and H. sapiens: To depict the difference in the average abundance of amino acids at different positions of the PTS1 in fish (A) and humans (B), we selected proteins from each species, which were predicted to harbour a PTS1 (Neuberger et al., 2003), and depicted them as WebLogo (http://weblogo.threeplusone.com/create.cgi). (C) A comparison of the numerical values of the PTS1-predictions for those proteins is plotted for H. sapiens (x-axis) and D. rerio (y-axis) and depicted relative to the threshold for the evaluation of targeted proteins (>0, red) or the “twilight zone” (–10 < x < 0, grey); proteins with negative prediction are highlighted. (D) Systematic overview of orthologous fish proteins, which were either not found in D. rerio (ZADH2/PTGR3) or lack a PTS1 (Usp-L, long isoform; Prdx-5). (+) or (–) indicate presence or absence of the protein; 2x indicates 2 variants; traditional PTS1 identified (green) or not found (red); non-canonical PTS1 (yellow). (E) Schematic representation of the two isoforms of ubiquitin carboxyl-terminal hydrolase 2 (USP2) in H. sapiens (blue) and D. rerio (red). No PTS1 was found in the long isoform of Usp2 (Usp2-L). (F) Schematic representation of the two isoforms of peroxiredoxin-5 (Prdx-5) in H. sapiens (green) and D. rerio (orange). The long variant harbours an additional mitochondrial targeting signal, whereas the C-terminal sequence is identical. (G,H) Sequence comparison of the C-terminal end of USP2 orthologues (G) and PRDX5-orthologues (H). The loss of the PTS1 in Usp2-L is unique for D. rerio, whereas the PTS1 of human PRDX5 is found only in more ancient fish species.

Verification of PTS1 functionality for selected candidate proteins. COS-7 cells were transfected with an EGFP fusion protein containing the C-terminal putative PTS1 of D. rerio Cdc5l DLLMLDKQTLSSKI(A), D. rerio Kctd5a DLKAKILQEQGSRM(B), and H. sapiens CDC5L DLLLEKETLKSKF(C). Cells were processed for immunofluorescence microscopy using anti-PMP70 (red) as a peroxisomal marker. White arrows highlight peroxisomes that are clearly detectable in both channels. Bars, 10 μm (A,C), 20 μm (B).

Localisation of HsCDC5L and HsKCTD5 in COS-7 cells. COS-7 cells were transfected with Myc-CDC5L (A–C) or Myc-KCTD5 (D–F) and processed for immunofluorescence microscopy using anti-Myc and anti-PEX14 (peroxisomal marker) antibodies. Note that a peroxisomal localisation is not detected (arrows), even at large peroxisomal structures (circles). Bars, 10 μm.