Peroxisome biogenesis, membrane contact sites, and quality control

Review10 December 2018free access Peroxisome biogenesis, membrane contact sites, and quality control Jean-Claude Farré Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA Search for more papers by this author Shanmuga S Mahalingam Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA Search for more papers by this author Marco Proietto Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA Search for more papers by this author Suresh Subramani Corresponding Author [email protected] orcid.org/0000-0003-0180-1742 Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA Search for more papers by this author Jean-Claude Farré Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA Search for more papers by this author Shanmuga S Mahalingam Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA Search for more papers by this author Marco Proietto Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA Search for more papers by this author Suresh Subramani Corresponding Author [email protected] orcid.org/0000-0003-0180-1742 Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA Search for more papers by this author Author Information Jean-Claude Farré1, Shanmuga S Mahalingam1, Marco Proietto1 and Suresh Subramani *,1 1Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, USA *Corresponding author. Tel: +1 858 534 2327; E-mail: [email protected] EMBO Rep (2019)20:e46864https://doi.org/10.15252/embr.201846864 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues. Glossary aa amino acid ACBD acyl-CoA binding domain ADP adenosine diphosphate APX ascorbate peroxidase Arf ADP-ribosylation factors ATPase adenosine triphosphatase BAK BCL2 antagonist/killer BiFC bimolecular fluorescence complementation CAML calcium-modulating cyclophilin ligand cAMP cyclic adenosine monophosphate Cat carnitine transferase CAT catalase CERT ceramide transfer protein CHO Chinese hamster ovary Cit citrate synthase CTD C-terminal domain Cys cysteine DAG diacylglycerol DHA docosahexaenoic acid Dnm dynamin DRP dynamin-related protein ERAD endoplasmic reticulum-associated degradation ER endoplasmic reticulum ERMES endoplasmic reticulum-mitochondrial encounter structures ERppVs endoplasmic reticulum-derived pre-peroxisomal vesicles ESCRT endosomal sorting complexes required for transport Fis fission GET guided entry of tail-anchor GFP green fluorescent protein GTPase guanosine triphosphatase GTP guanosine triphosphate HEK human embryonic kidney cells HSP high-speed pelletable ICL isocitrate lyase Inp inheritance of peroxisomes LD lipid droplet LPMC lysosome–peroxisome membrane contacts LSP low-speed pelletable MCS membrane contact sites MCTP2 multiple C2 domain containing transmembrane protein MDppVs mitochondrially derived pre-peroxisomal vesicles MFF mitochondrial fission factor mPTS membrane peroxisomal targeting signals MTS mitochondrial targeting signal Myo myosin NTD N-terminal domain OSBP oxysterol binding protein PBDs peroxisome biogenesis disorders PE phosphatidylethanolamine pER pre-peroxisomal endoplasmic reticulum Pex/PEX peroxins from yeast/mammals Phe phenylalanine PMPs peroxisomal membrane proteins Pp Pichia pastoris ppVs pre-peroxisomal vesicles Psd phosphatidylserine decarboxylases PS phosphatidylserine PTS peroxisomal targeting signals QC quality control RADAR receptor accumulation and degradation in the absence of recycling RHD reticulon homology domain RING really interesting gene ROS reactive oxygen species Sc Saccharomyces cerevisiae SRP signal recognition particle TA tail-anchored TMD transmembrane domain TOMM20 translocator of outer mitochondrial membrane 20 TRC transmembrane recognition complex Ub ubiquitin UPS ubiquitin–proteasome system VAMP vesicle-associated membrane protein VAP VAMP-associated protein VDAC voltage-dependent anion channel WRB tryptophan-rich basic protein WT wild type YFP yellow fluorescent protein Introduction Peroxisomes are a conserved, intracellular organelle of eukaryotic cells and are involved in a range of metabolic functions that vary based on the organism in which they occur. General functions of metabolic pathways housed in peroxisomes include the β-oxidation of fatty acids and the detoxification, by catalase, of hydrogen peroxide that is produced during fatty acid oxidation 1. Other metabolic functions and the role of peroxisomes in human disease are reviewed elsewhere 2, 3. A characteristic feature of peroxisomes is that they proliferate or dissipate in response to external cues 4. In yeasts, peroxisome numbers, sizes, and enzyme repertoires can rapidly change by manipulating the carbon source in their growth medium. For example, Saccharomyces cerevisiae and Pichia pastoris will proliferate peroxisomes when grown in fatty acids, such as oleate, because the β-oxidation of fatty acids occurs in peroxisomes. P. pastoris and Hansenula polymorpha also proliferate peroxisomes when grown in methanol, which is metabolized using peroxisomal enzymes. Conversely, when organisms are switched from peroxisome proliferation conditions to media that do not require peroxisomal metabolism, then the excess peroxisomes are degraded, typically by a selective form of autophagy called pexophagy 5. Similarly, excessive reactive oxygen species (ROS), hypoxia, or the depletion of iron can trigger pexophagy in different model organisms 6-9. The proteins implicated in peroxisome biogenesis are known as peroxins and the genes encoding them are dubbed PEX genes. More than half of these peroxins, referred to as Pex or PEX proteins in yeast and mammals, respectively, are required for the import of peroxisomal matrix proteins, and the rest are implicated in the targeting of the peroxisomal membrane proteins (PMPs) to the peroxisome membrane and in peroxisome proliferation. This review will mostly focus on exciting, new advances regarding peroxisome biogenesis, membrane contact sites (MCS) between peroxisomes and other organelles, and quality control (QC), while only a brief description of peroxisomal matrix import is provided for continuity. These topics will highlight the flexibility exploited by different model organisms in the relative use of redundant pathways for PMP and peroxisome biogenesis, the interconnectivity and communication between peroxisomes and other subcellular compartments, and the complex QC mechanisms associated with peroxisomes. Brief overview of peroxisomal matrix protein import Proteins destined for import into the peroxisome matrix or membrane possess peroxisomal targeting signals (PTSs) or membrane PTSs (mPTSs), respectively (Fig 1). The peroxisomal matrix proteins are synthesized in the cytosol and transported into the peroxisome matrix across translocons located in the peroxisome membrane (Fig 1A). Most peroxisomal matrix proteins have either a C-terminal PTS1 or an N-terminal PTS2. In yeast and mammals, these sequences are recognized by specific receptors, Pex5, for PTS1 and Pex7 for PTS2. The protein Pex9 is a Pex5-related protein found in S. cerevisiae that acts on limited PTS1 cargos, such as malate synthase 1 and 2, as well as the glutathione transferase, making it a condition-specific PTS receptor 10, 11. These receptors can either act alone (e.g., Pex5), or with co-receptors (Pex7-Pex18 or Pex7-Pex20 in S. cerevisiae and P. pastoris, respectively, or PEX7-PEX5L in mammals) to form receptor/cargo complexes, which dock at the peroxisome membrane with a docking complex (typically comprised in yeasts of Pex13, Pex14, and Pex17, but mammals lack Pex17). The minimal translocon in yeast involves Pex14 and Pex5 for PTS1 import 12, and likely Pex14/Pex17 and Pex18 for PTS2 import 13. Associated with the docking complex is another subcomplex comprised of three conserved RING (really interesting gene) domain proteins, Pex2, Pex10, and Pex12, that have E3 ligase activities. Together, the docking and RING subcomplexes form the importomer complex 14, 15. Figure 1. Peroxisomal matrix and membrane protein import in yeast (an overview)Most proteins destined for import into the peroxisome matrix possess either a C-terminal PTS1 or an N-terminal PTS2. (A) The peroxisomal matrix protein import cycle. These cargos synthesized in the cytosol are recognized by PTS receptors, Pex5 for PTS1 and Pex7 for PTS2, respectively. Pex7 generally works with a co-receptor (Pex18/21 in S. cerevisiae or Pex20 in P. pastoris only Pex18 is shown). Pex9 is a Pex5-related protein found in S. cerevisiae that acts on limited PTS1 cargos as described in the text 10, 11. The PTS receptor/cargo complex, along with the co-receptor, where applicable, docks at the peroxisome membrane with the docking complex, comprised of Pex13, Pex14, and Pex17. PTS cargos are translocated into the peroxisome matrix across translocons in the peroxisome membrane. The minimal translocon in yeast involves Pex14 and Pex5 for PTS1 import 12, and likely Pex14/Pex17 and Pex18 for PTS2 import 13. Associated with the docking complex is the RING subcomplex comprised of Pex2, Pex10, and Pex12 that have E3 ligase activities involved in ubiquitin-dependent, PTS-receptor recycling and QC steps (sections Brief overview of peroxisomal matrix protein import and QC during peroxisomal matrix protein import). Together, the docking and RING subcomplexes form the importomer complex 14, 15. Following PTS cargo release in the peroxisome lumen, the PTS receptors, and co-receptors where applicable, recycle from the peroxisomes back to the cytosol for another round of import, using components collectively called the exportomer, whose components are described in the text 16. (B) The PMP import cycle for the direct import of proteins into the peroxisome membrane (section The direct import of PMPs to peroxisomes). Each PMP has at least one mPTS that is bound to, and the PMP is chaperoned by, Pex19, which docks at the peroxisomes via interactions with Pex3. The PMP is inserted into the membrane and Pex19 recycles back to the cytosol for another round of PMP import. Download figure Download PowerPoint The receptor/cargo complexes from the cytosol interact with the docking subcomplex, translocate into the peroxisome matrix or membrane and release their respective cargos in the peroxisome lumen. Then, the receptors, and co-receptors where applicable, recycle from the peroxisomes back to the cytosol for another round of import, using components collectively called the exportomer 16, 17. This export and recycling of the receptor and co-receptor requires mono-ubiquitination of a cysteine near the N-terminus of Pex5 (in yeast and mammalian systems) 18, 19 and Pex20 (in P. pastoris) 20. Pex5 and Pex20 mono-ubiquitination requires the typical ubiquitination enzymes—an E1 protein, an E2 in the form of Pex4 associated in yeast with the peroxisome membrane via the PMP, Pex22, and E3 ligase activity provided by one or more components of the peroxisomal RING subcomplex 21. The mono-ubiquitinated PTS receptors or co-receptors are recognized by peroxisome membrane-associated AAA-ATPases, Pex1 and Pex6 22, 23, which are associated with peroxisomes in an ATP-dependent manner via interaction with specific PMPs (Pex15 in yeast or PEX26 in mammals). These ATPases are required to export and recycle mono-ubiquitinated PTS receptors/co-receptors 17, following which the PTS receptors/co-receptors are deubiquitinated (by Ubp15 for the mono-ubiquitinated Pex5 in yeast or by USP9X in mammals) and reused for subsequent rounds of import 24, 25. When this mono-ubiquitination is blocked, either by mutation of the ubiquitination site in the exported receptor or co-receptor or by mutations in the receptor recycling machinery that recognizes this mono-ubiquitin and exports the proteins to the cytosol, then an alternative pathway called receptor accumulation and degradation in the absence of recycling (RADAR) takes over 20. This is described later under quality control pathways. Peroxisomal membrane proteins Because many pex mutants (with the exception of pex3, pex16, and pex19) are defective only in peroxisome matrix protein import and still possess peroxisome remnants or ghosts containing PMPs, the sorting of PMPs requires components distinct from those involved in peroxisomal matrix protein import. PMPs fulfill a variety of functions such as serving as components of the peroxisomal translocon or the exportomer, membrane transporters for metabolites and ions, quality control or organelle division machineries, redox proteins, signaling molecules, organelle membrane tethers, and so on. PMPs have one or more mPTSs 26 that are sorted to the peroxisome membrane in either a Pex19-dependent or Pex19-independent manner 27. Based on whether or not Pex19 is required for their membrane insertion step, these PMPs are broadly classified into two classes: Class I or direct pathway—involving Pex19-dependent membrane insertion of PMPs (most PMPs) 28-30 (Fig 1B). Class II or indirect pathway—this alternative was proposed initially to address the PEX19-independent membrane insertion of PEX3 in mammalian cells 30, 31, which traffics to peroxisomes via the endoplasmic reticulum (ER) 32, 33. However, since these early studies, many PMPs have been shown to traffic to the peroxisomes via the ER, we therefore prefer to call this the indirect pathway (i.e., via the ER) of PMP trafficking to peroxisomes 34. It should also be noted that the same PMP may traffic to peroxisomes directly or indirectly. Thus, mammalian PEX3 can also be imported directly to peroxisomes in a PEX16- and PEX19-dependent manner 35. Perhaps many (or even all) PMPs have the flexibility to be targeted to peroxisomes directly, or indirectly via the ER 34, with the latter being the only mode possible when there are no pre-existing peroxisomes. A subclass of PMPs is the tail-anchored (TA) proteins—integral membrane proteins with a short, C-terminal sequence adjoining their transmembrane domain (TMD) 36, whose insertion into the membranes (peroxisomal or ER) may be Pex19-dependent or Pex19-independent, either directly into pre-existing peroxisomes or indirectly via prior insertion into membranes of other subcellular compartments, from which peroxisomes are subsequently derived. These topics are addressed later (sections The direct import of tail-anchored proteins to peroxisomes and Insertion of tail-anchored PMPs into the ER membrane). Peroxisome biogenesis—divergent models ranging from growth and division to de novo mechanisms Two models have co-existed for decades regarding the biogenesis of peroxisomes and are likely to operate within the same cells in response to specific environmental cues. The older of these is the growth and division model 37, in which peroxisomes, like chloroplasts and mitochondria, arise from pre-existing peroxisomes that grow to a certain size after acquiring their PMPs and matrix proteins directly from the cytosol. Then, upon activation by poorly characterized mechanisms, peroxisomes divide by fission to form a daughter peroxisome that then goes through this cycle again. The second model invokes de novo peroxisome biogenesis in which some PMPs are first inserted into the membrane of the ER, sorted to a region of the ER called the pre-peroxisomal ER (pER), from where distinct pre-peroxisomal vesicles (ppVs) containing the PMPs bud 38. Moreover, a recent study in mammals suggests that some ppVs might also originate from the mitochondria 39. The ppVs containing different subsets of PMPs then fuse, either in a heterotypic fashion 40 or with pre-existing peroxisomes 41 to create mature or larger peroxisomes, respectively. Finally, a third model blends and accommodates features of the PMP traffic envisioned in the growth and division model, as well as via the ER in the de novo biogenesis model 42. This third model invokes two routes for PMP insertion into peroxisomes—one involving direct insertion of PMPs into membranes of pre-existing peroxisomes and the other invoking indirect traffic of PMPs to peroxisomes via the ER/mitochondria, followed by their subsequent sorting to the peroxisomes 37-39. In this review, we will mostly focus on the first two models, although the indirect PMP traffic via the ER invoked in this third model will be described in some detail in the de novo peroxisome biogenesis model (section The de novo peroxisome biogenesis model). The growth and division model The direct import of PMPs to peroxisomes In the growth and division model, PMPs are inserted directly into the peroxisome membrane from the cytosol and the ER provides the lipids for membrane growth, most likely through organelle contact sites described later (section Peroxisome-ER MCS) 41. PMPs are synthesized on free polyribosomes and post-translationally imported into peroxisomes. Their hydrophobic TMDs have to be protected by chaperones soon after synthesis. Their mPTSs consist of a cluster of basic residues in a predicted α-helical conformation with a minimal length of 11 amino acids and are generally flanked by one or two TMDs 43. Pex19 is an acidic peroxin that associates with membranes through its C-terminal farnesyl tail, and serves as a receptor and chaperone for Class I PMPs, recognizing and binding the mPTSs within these PMPs 28, 29. The binding of Pex19 near the TMDs of such PMPs facilitates the role of Pex19 as a chaperone 30. This role of Pex19 in stabilizing and chaperoning hydrophobic PMPs is underscored by the fact that several PMPs are unstable and degraded in cells lacking Pex19 44. Furthermore, the solubility of in vitro synthesized PMPs, such as PMP22, increases in the presence of Pex19 45. Pex19 is a predominantly cytosolic protein that exhibits a characteristic domain organization 27, 46. A small but significant amount of the Pex19 population is also associated with the peroxisome membrane through the farnesylation of its C-terminal end 47. The C-terminal domain (CTD) of Pex19 participates in the recognition and binding of mPTS motifs in PMPs 48-50. A role for the farnesylation of Pex19 is still unclear. Pex19 does not seem to require farnesylation to associate with membranes, and there are reports that it functions to allosterically modulate Pex19 function 51. Nuclear magnetic resonance data suggest that the C-terminal residues of the CTD become rigid upon farnesylation, which in turn, might enhance the interactions of mammalian PEX19 with PMPs 51. In rats and mice, a splice variant of PEX19, called PEX19i, has been identified, which encodes a PEX19-like protein with its C-terminal farnesyl tail replaced by a hydrophobic region 52. The transcription of PEX19i was highly induced by the peroxisome proliferator, clofibrate, and this protein was functional in that it restored peroxisomes by complementation of PEX19-deficient (ZP119) Chinese hamster ovary (CHO) cells and also bound several PMPs known to interact with PEX19. The ability of this protein to support peroxisome biogenesis also suggests that the farnesylation of PEX19 is not critical for its function. Both Pex3 and Pex19 are involved in membrane insertion of the PMPs 53, 54. Pex19 directs the PMP to the peroxisomal membrane, where it docks with the transmembrane protein, Pex3, and thereby acts as a shuttling receptor (Fig 1B) 55. Surprisingly, only these two factors, Pex3 and Pex19, seem to be essential for the Class I pathway, independent of the topological complexity of the PMPs. It has been shown in mammals and Neurospora crassa that PMPs harboring one to six TMDs can be inserted into peroxisome membranes through this route 53, 56. The N-terminal region of Pex19 contains a high-affinity Pex3-binding site 48, 50, 55, 57. Pex3 possesses one TMD near its N-terminus and exposes most of its polypeptide chain into the cytosol 58-60. The cytosolic domain of Pex3 serves as a docking factor for Pex19-PMP complexes 55. Because lipid molecules can bind to Pex3 in competition with Pex19, such lipid binding may perturb the peroxisomal lipid bilayer to allow PMP insertion into the peroxisome membrane 61. The Pex3 mPTS does not bind Pex19 directly, and therefore, its membrane insertion follows the indirect pathway 30, trafficking through the ER and possibly mitochondria (section ppVs derived from mitochondria (mammals)), rather than by direct import into peroxisome membranes. Nevertheless, mammalian PEX3 traffic to the peroxisome membrane depends on PEX16, which is a Class I PMP itself, and might also serve as a docking factor for PEX3-PEX19 complexes at the peroxisome surface under conditions when PEX3 is forced to traffic to peroxisomes using the Class I pathway 35. The direct import of tail-anchored proteins to peroxisomes At least two proteins implicated in peroxisome biogenesis in mammals are the TA PMPs (FIS1 and PEX26), which can be inserted directly by the Class I pathway 53. Evidence of the direct targeting of PEX26 in mammalian cells comes from a cell-free reaction in which a complex containing PEX19 and PEX26 accumulates in a pex3 mutant cell line, and PEX26 from this complex can be targeted to peroxisomes in semi-permeabilized cells in a PEX3-dependent, but ASNA1/TRC40-independent, manner 62. Either removal of the mPTS in PEX26 or the absence of PEX19 in mammalian cells impairs PEX26 targeting to peroxisomes. A ternary complex between PEX19, PEX26, and PEX3 has been detected 54. The yeast orthologue of PEX26 is Pex15 and it too is targeted to peroxisomes in a similar manner 63. Interestingly, a new function has been uncovered for Pex19 in S. cerevisiae, which is also apparently involved in the insertion of the TA proteins, Fis1 and Gem1, into mitochondria 64. How is TMD binding and release mediated during direct insertion of PMPs into the peroxisome membrane? This role of Pex19 was addressed using Neurospora proteins 53. Pex19 was reported to bind Pex26, preventing it from aggregation followed by its insertion into peroxisome membranes in a Pex3-dependent manner, mimicking the mammalian system. This chaperone-like activity of Pex19 depends on hydrophobic contacts via an amphipathic helix in the CTD of Pex19 and the TA PMP. This study also identified an additional amphipathic helix in Pex19, lying between the N-terminal, Pex3 binding region in Pex19, and its CTD. Hydrophobicity in this region of Pex19 is obligatory for the insertion of the TMD of the TA PMP, but not for chaperone activity or Pex3 binding. Another hydrophobic surface at the base of Pex3, adjacent to where it is anchored in the membrane, promotes an unconventional form of membrane association of the TA PMP and is also required for the membrane insertion of its TMD. Together, these data support a model in which hydrophobic moieties in Pex19 and Pex3 act in distinct capacities to promote TMD binding, release, and insertion. However, PEX26 and its yeast orthologue, Pex15, are capable of also targeting to peroxisomes via the ER (probably a minor pathway) in mammalian cells, in what is reminiscent of the indirect pathway 65. This pathway is described later (section Insertion of tail-anchored PMPs into the ER membrane). Peroxisome fission in the growth and division model According to the current model, during peroxisome growth and division, peroxisome fission happens in a 3-step process involving peroxisome elongation, constriction, and scission (Fig 2, top panel) 41. Pex11 is essential for the first step (Fig 2, panel 1). Its overexpression causes peroxisome proliferation, and its deletion causes enlarged peroxisomes and a decrease in their number 66. Penicillium chrysogenum Pex11 was shown in vitro to bind, impart curvature, and tubulate liposome membranes, particularly those containing negatively charged phospholipids mimicking those in peroxisome membranes 67. This feature is conserved from yeast to human PEX11 isoforms. Figure 2. Peroxisome fission in the growth and division modelAccording to the growth and division model, peroxisome fission happens in a 3-step process. During the first step of elongation, Pex11 (PEX11β in mammals), a transmembrane protein that imparts curvature to peroxisome membranes (panel 1), is essential for the elongation step. The topology shown here for Pex11 is based on studies in H. polymorpha 67. The second step, involving membrane constriction, is poorly understood and we do not know any proteins implicated in this step. The third step, peroxisome fission, starts in P. pastoris with the phosphorylation of Pex11(S173) that stimulates its interaction with the adaptor, Fis1 (panel 2) 78. Note that the topology of PpPex11 has not been documented, so it is unclear whether the phosphorylation is on the cytosolic or the peroxisome matrix side. Fis1 then recruits the peripheral receptors, Mdv1 and/or Caf4 (panel 3) 75. Mdv1 and/or Caf4 assemble a Dnm1 ring around the peroxisome constriction site (panel 4). Mammals do not have homologues for these proteins, and DRP1 is recruited to peroxisomes by MFF and FIS1 79. Yeast Dnm1 interacts with Fis1 and two Pex11 helices named B1 and B3 (panel 5). The hydrolysis of GTP by Dnm1, enhanced by the interaction with the B3 helix of Pex11, leads to a constriction that divides the peroxisome 82. Download figure Download PowerPoint While Pex11 causes membrane tubulation in vitro, the cytoskeleton to which peroxisomes are attached in yeast and mammalian cells likely also plays a role in peroxisome tubulation and elongation, prior to division. Yeast peroxisomes are associated with an actin/myosin cytoskeleton, involving the Myo2 motor linked to peroxisomes via the proteins, Inp1 and Inp2 (inheritance of peroxisomes) 68, 69. In mammals, however, peroxisomes are associated with microtubules through the Ras GTPase, MIRO1, a potential adaptor linking mammalian peroxisomes to microtubules 70. MIRO1 localizes to both peroxisome and mitochondria. Distinct splice variants of MIRO1 are targeted specifically to peroxisomes and mitochondria in human embryonic kidney (HEK) cells, with the MIRO1-variant 4 being more specific for peroxisomes in these cells. When MIRO1 is targeted exclusively to peroxisomes, it mediates pulling forces that contribute to peroxisome membrane elongation and proliferation in a cell type-dependent manner 70. It should be noted, however, that in mammalian cells, peroxisomes can also elongate independently of microtubules, and peroxisome elongation is promoted by microtubule-depolymerizing drugs 71, 72. This suggests that a PEX11 isoform, PEX11β, and motor forces such as those mediated by MIRO1 can independently promote peroxisome proliferation, but may cooperate under physiological conditions. There is less information about peroxisome constriction, and actually, this step is poorly understood. However, given the fact that peroxisomes share their components of a common division machinery with mitochondria, some insights may be gleaned from the multiple constriction steps involved in mitochondrial division 73, 74. In S. cerevisiae, the GTPase, Dnm1, accomplishes the final step of scission 75. In dnm1Δ cells, a single enlarged peroxisome protrudes from the mother cell into the bud, demonstrating that Dnm1 is required for the final step (scission), but not for the elongation step 76. Dnm1 forms a ring-like structure around membranes, and the hydrolysis of GTP leads to a constriction that divides the organelle 77 (Fig 2, panel 4). Unlike canonical dynamins, yeast Dnm1 does not have pleckstrin-homology domains for direct membrane binding. Instead, it binds to adaptors, such as Fis1, a TA protein localized to both peroxisomes and mitochondria 75. Fis1 interacts with phosphorylated Pex11 (as described later in this section) at peroxisome membranes 78 and recruits the yeast peripheral membrane receptors, Mdv1 and Caf4, which, in turn, assemble Dnm1 75 (Fig 2, panel 2–4). In higher eukaryotes that do not have Mdv1 and Caf4 ho