1995;11:395C401. 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with genes essential for peroxisome assembly have been isolated (for reviews, see Distel and encode peroxisome targeting signal (PTS)-1 and PTS-2 receptors, respectively. and are thought to encode the receptor docking proteins on the peroxisomal membrane, ZM39923 STMY based on their ability to ZM39923 bind PTS receptors. and are proposed to be essential for the proper localization and stability of peroxisomal membrane proteins (Honsho gene products, especially for membrane dynamics, are still unknown. and encode AAA family ATPases (Kunau and the fusion was reconstituted in vitro (Titorenko suggested that Pex6p and Pex1p are involved in the late steps of peroxisome biogenesis (Collins (1988a , 1988b ) reported that fibroblasts (GM 4340) derived from a mutants are in the import of the matrix proteins across the peroxisomal membrane. Knowledge about the morphology of the peroxisomal remnant structures (peroxisomal ghosts) in or mutants is still limited, especially about their membrane morphology. Another point to be clarified ZM39923 is whether peroxisomes were assembled from the ghosts during the genetic complementation. We presented indirect evidence that the preexisting ghosts serve as precursors of peroxisomes at least in a gene. MATERIALS AND METHODS DNA Constructs phGFP(105)-C1 (pGFP) was ZM39923 used as a GFP expression vector (Yamasaki cDNA expression plasmid was pUcD292A (Tsukamoto cDNA, a microscope (IMT-2) using an oil immersion objective lens (SPlanApo 60, NA = 1.4) and high-selectivity filters for GFP and Cy3 (cDNA The cell suspension (200 l, 5 107 cells/ml) was mixed with 10 g of pUcD292A and electroporated (Model BT-600; BTX, San Diego, CA). A cuvette with electrodes 2 mm apart was used, and the settings were 110 V, 3100 F, and 72 . Cells from two electroporation cuvettes were mixed and plated into four 60-mm dishes, cultured for 1, 4, 8 and 24 h, and then used for EM. To intensify signals of catalase histochemistry, 5 107 cells/ml ZP92 were transfected with 30 g of pCatalaseSKL, 20 g of ptet-On ((Palo Alto, CA). Autoradiographic images were taken by an imaging plate and quantified by BAS 2000 or FLA 3000 (Fuji Film). Electron Microscopic Cytochemistry Cells were fixed for 1 h at room temperature with 2% glutaraldehyde in 0.1 M HEPES/NaOH (pH 7.4). Cells were postfixed with 1% reduced osmium tetroxide for 1 h at room temperature, dehydrated in ethanol and propylene oxide, and embedded in Epon 812. Thin sections on copper grids were briefly contrasted with uranyl acetate and lead citrate before examination. For cytochemistry of catalase, fixed cells were incubated for 2 h at 37C in medium containing 2 mg/ml DAB, 0.1 M glycine buffer (pH 10.5), and 0.02% H2O2 before postfixation, and uranyl acetate staining was omitted. Immunoelectron Microscopy For postembedding immunoelectron microscopy (immuno-EM), cells were fixed for 1 h at room temperature with 4% formaldehyde and 0.25% glutaraldehyde in 0.1 M HEPES/NaOH (pH 7.4). After rapid dehydration in ethanol, they were embedded in L. R. White. Thin sections on nickel grids were preincubated on a drop of 0.5% BSA in PBS. The sections were incubated overnight in the 1/1000 diluted primary antibody. ZM39923 After being rinsed with PBS, the sections were incubated for 30 min on a drop of protein A-gold (15 nm) prepared as described (De Roe for 10 min. The PNS fraction was divided into three portions. One part of the PNS was centrifuged for 20 min at 18,500 knockout mouse (Baes cDNA Next, we analyzed whether the complex membrane structures become import competent.