Research ArticleFis1, DLP1, and Pex11p coordinately regulate peroxisome morphogenesis
Introduction
Peroxisome is a single-membrane-bounded, ubiquitous intracellular organelle present in a wide variety of eukaryotic cells from yeast to humans. The function of peroxisome includes the metabolism of various organic compounds such as β-oxidation of very long chain fatty acids and synthesis of plasmalogens, cholesterol, and bile acids [1]. The physiological importance of this organelle is highlighted by the peroxisome biogenesis disorders (PBD), including Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and rhizomelic chondrodysplasia punctata, caused by the congenital deficiency of peroxisome biogenesis. Patients with PBD are classified into 13 different genotypes [2], [3], [4]. Peroxisome-deficient cell mutants, including PBD patients-derived fibroblasts as well as Chinese hamster ovary (CHO) and yeast cells, have made it possible to genetically identify and isolate the essential PEX genes encoding peroxins [3], [5], [6], [7]. Therefore, peroxisome has been extensively used as a useful model for investigating the protein traffic, biogenesis of intracellular organelles, and human protein kinesis disorders [5], [6], [7], [8], [9], [10], [11].
Over 30 peroxins, including 14 in mammals, have been cloned. Of the mammalian Pex11p isoforms, Pex11pα, Pex11pβ, and Pex11pγ, the constitutively expressed Pex11pβ is better known to induce peroxisome proliferation [12], [13]. Higher level expression of Pex11pβ promotes peroxisome division in mammalian cells [13], [14], whereas PEX11β null mice show the reduced peroxisome number in cells, manifesting the phenotype seen in patients with PBD despite of abrogating peroxisome function [15]. These findings indicate that Pex11pβ plays important roles in peroxisome morphogenesis. Dynamin-like protein 1 (DLP1) essential for mitochondria division was recently reported to be also involved in peroxisome division [16], [17], [18]. However, the molecular mechanisms of Pex11pβ- and DLP1-mediated peroxisome division remained little understood. Fis1 was reported as a key regulator of mitochondrial morphology [19], [20], [21]. More recently, Fis1 was shown to be involved in peroxisome fission as well [22].
We here identified Fis1 as the third factor responsible for peroxisome morphogenesis. We also provide several lines of evidence for the ternary complexes comprising Pex11pβ, Fis1, and DLP1 and discuss their functional consequences.
Section snippets
Antibodies
We used rabbit antisera to peroxisome matrix targeting signal type 1 (PTS1) [23], Pex14p [24], rat acyl-CoA oxidase (AOx) [25], malate dehydrogenase (MDH) [26], human Fis1 (Apotech), and hemagglutinin A (HA) [27]; mouse monoclonal antibodies to Myc (9E10) and human Tom20 (F-10) (Santa Cruz Biotech), rat DRP1 (Transduction Lab.), and chicken tubulin-α (Seikagaku Kogyo, Tokyo, Japan); guinea pig antibody to rat Pex14p [28].
Cell culture
CHO cell lines including wild-type CHO-K1 and dlp1 ZP121 [18], [29] were
Fis1 is localized to mitochondria and peroxisomes
DLP1, an essential factor for mitochondrial division, was recently shown to be involved in peroxisomal division [16], [17], [18]. DLP1 is recruited to mitochondria from the cytosol by Fis1, a regulator of mitochondria morphology [19], [20]. To verify whether Fis1 is localized to peroxisomes, we stained wild-type CHO-K1 cells with anti-Fis1 antibody. Fis1-positive punctate structures were detected, mostly superimposable with those visualized with MitoTracker (not shown), indicative of
Fis1 is involved in peroxisomal morphogenesis
DLP1 is required for the mitochondria division [38], being recruited to mitochondria by Fis1 from the cytosol [19], [20], [21]. Recently, DLP1 was also shown to be involved in the peroxisome division [16], [17], [18]. In the present work, we found that endogenous Fis1 was also localized to peroxisomes in addition to mitochondria in CHO-K1 cells, consistent with the recent report describing Fis1 in peroxisomes purified from rat liver [22]. Peroxisomal localization of Fis1 was more evident in
Acknowledgments
We thank T. Matsuzaki for the instruction of mammalian two-hybrid assay, M. Nishi for preparing figures, and the other members of our laboratory for discussion. This work was supported in part by SORST grant (to Y.F.) from the Science and Technology Agency of Japan; Grants-in-Aid for Scientific Research (to Y.F.), Grant of National Project on Protein Structural and Functional Analyses (to Y.F.), and The 21st Century COE Program from The Ministry of Education, Culture, Sports, Science, and
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