| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2000: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Peroxisomes are ubiquitous organelles in eukaryotic cells and surrounded by a single membrane. Several peroxisome biogenesis disorders have been discovered and studied. By genetic complementation using peroxisome-deficient mutants of yeast and mammalian cells, at least 23 PEX genes essential for peroxisome assembly have been isolated. However, the functions and functional mechanisms of most PEX gene products, especially for membrane dynamics, are still unknown. 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 cCDNA, 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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