| Project/Area Number |
08660097
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
HIRATA Aiko The University of Tokyo, Institute of molecular and Cellular Biosciences, Instructor, 分子細胞生物学研究所, 助手 (30092307)
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1997: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | immno-electron microscopy / ultrastructure / budding yeast (S accharomyces cerevisiae) / secretory pathway / ER (endoplasmic reticulum) / heterologous protein / membrane protein / 酵母 / 分裂酵母 / 液胞 |
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| Research Abstract |
Yeasts have been studied as a model system of eukaryotic cell for the genetic analysis, but regarded not promising for ultrastucture studies. Recently rapid freezing and substitution method has improved greatly the fixation not only of ultrastructure of yeast cell but also of immunoactvity of the proteins, which enabled to localize the proteins relative to the ultrastucture.
We compared the results studied by immuno-electron microscopy using different substitution methods to localize the proteins with respect to the ultrastucture in the yeast Saccharomyces cerevisiae.
(1) Cells were rapidly fixed into liquid propane cooled with liquid nitrogen and substituted with 4%O_sO4/acetone (-80ﾟC) and embedded in Spurr's resin. Sections were treated with anti aspartic proteinase antibody and then with colloidal gold-conjugated anti-rabbit IgG antibody. The material was Saccharomyces cerevisiae in which heterologous prosequence-deleted derivative of an aspartic proteinase from a filamentous fungus
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Rhizopus niveus was expressed. Immuno-electron microscopical analysis revealed that the DELTApro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope.
(2) Cells were substituted with ethanol after rapid frozen and polymerized in LR White resin. Sections were treated with anti-myc antibody and then with colloidal gold-conjugated anti-mouse IgG antibody. We isolated mutants defective in glycosylation of the outer chain to investigate glycosylation mechanism in the Golgi . Vanadate-resistant and immature glycosylation mutants belonged to nine complementation groups. One of them was VRG4 which encoded a Golgi GDP-mannose transporter (a transmembrane protein). The material was S.cerevisiae in which the myc-tagged Vrg4 protein was overproduced. These results showed that the Golgi and ER membranes proliferated. Some of these membranes showed the stacked structures like in mammalian cells. At the same time, immunogold labeling revealed that the overproduced Vrg4-myc proteins localized in such proliferated membrane structures.
In both studies, cells were showed a good preservation of ultrastructure and immno activity of proteins. Less
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