Abstract
ABSTRACTWhen Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast.
Highlights
Appearance of fluorescent inclusion bodies in elongated mitochondria When the 3626 strain of S. cerevisiae that was cultured to the stationary phase was observed under UV excitation by fluorescence microscopy, needle-like structures with an average length of 4.6 mm that emitted autofluorescence were observed in almost all cells (Fig. 1A)
We referred to these structures as mitochondrial fluorescent inclusion bodies (MFIBs)
Immunoelectron microscopy with anti-Ald4p antibody showed that 10 nm gold-colloids localized to the filamentous inclusion body in both cross and longitudinal sections (Fig. 5E,F). These results indicated that the filamentous inclusion body revealed by electron microscopy coincided with MFIBs revealed by fluorescence microscopy, in which Ald4p is contained as a major component
Summary
Yeast mitochondria dynamically change their morphology depending on the life cycle stage of cells, as well as on. Changes in the number and morphology of mitochondria in S. cerevisiae have been extensively studied by electron microscopy of both vegetative and sporulating cells (Stevens, 1981). Morphological changes of mitochondria during meiosis and the sporulation process can be vitally visualized by double-staining of cells with DAPI and 3,39dihexyloxacarbocyanine iodide {DiOC6(3)} (Miyakawa et al, 1994)
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