Three-dimensional analysis of protein aggregate body in Saccharomyces cerevisiae cells

Modified genes of peroxisomal isocitrate lyase of Candida tropicalis (CT-IC L) were constructed and expressed in Saccharomyces cerevisiae cells. We obs erved subcellular localization of expressed products of the mutant CT-ICL g enes by immunoelectron microscopy. An unknown structure termed a protein ag gregate body (PAB) storing the expressed product was observed in cytoplasm in various mutants (Kamasawa ct al. (1996) J. Electron Microsc. 45:491-497) . We chose two typical cells harbouring the mutant ICL genes Delta 55Q and De lta 237-339 to analyse the ultrastructure and three-dimensional (3D) struct ure of PABs. The PABs had a homogeneous matrix with a wavy periphery in the cell image using a high-pressure freezing fixation method. Although PABs c ould not be separated from the cytoplasm or mitochondria under a confocal f luorescence microscope, 3D reconstruction of serial electron micrographs cl early showed the PAB was an independent structure of varying size and had t he shape of an incomplete sphere. A cell was sometimes observed to have mul tiple PABs.