Peroxisomes Are Formed from Complex Membrane Structures in PEX6-deficient CHO Cells upon Genetic Complementation

Noriyo Hashiguchi¹, Tomoko Kojidani¹, Tsuneo Imanaka², Tokuko Haraguchi³, Yasushi Hiraoka³, Evelene Baumgart⁴, Sadaki Yokota⁵, Toshiro Tsukamoto⁶^*, and Takashi Osumi¹

¹ Department of Life Science, Faculty of Science, Himeji Institute of Technology, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan
² Laboratory of Cell Biology, Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan
³ Kansai Advanced Research Center, Communications Research Laboratory and CREST of Japan Science and Technology Corporation, 588-2 Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan
⁴ Department of Anatomy and Cell Biology, Division of Medical Cell Biology, University of Heidelberg, Heidelberg, Germany (present address: Department of Anatomy and Cell Biology II, Justus Liebig University , Aulweg 123, D-35385 Giessen, Germany)
⁵ Yamanashi Medical University, 1110 Shimokatou, Tamaho, Yamanshi, 409-3898, Japan
⁶ Department of Life Science, Faculty of Science, Himeji Institute of Technology, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan (present address: Genomics Research Institute, Utsunomiya University, Mine-Machi 350, Utsunomiya, Tochigi, 321-8505, Japan)

^* Corresponding author. E-mail address: tsukamot{at}cc.utsunomiya-u.ac.jp.

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70 kD peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that: 1) Peroxisomal ghosts are complex membrane structures. 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.

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