| Project/Area Number |
05454648
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Cell biology
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
MIHARA Katsuyoshi Graduate School of Medical Science, Kyushu, University Associate Professor, 大学院・医学系研究科, 助教授 (40029963)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1994: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1993: ¥3,800,000 (Direct Cost: ¥3,800,000)
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| Keywords | Endoplasmic reticulum / Membrane protein / Membrane translocation / Vesicle transport / Retention signal |
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| Research Abstract |
ER retention signal of cytochrome P450 : To elucidate the determinants that cnable the P450s to be located in the ER,we constructed cDNAs encoding chimeric proteins in which an N-glycosylated secretory protein, carboxyesterase Sec, was connected to the N-terminus of the full-length, or truncated forms of a microsomal P450 and the constructs were expressed in COS cells. The subcellular localization of the chimeric proteins was determined by endo H sensitivity and immunofluorescence microscopy. The results with the fusion proteins indicated that transmembrane segemnt of P450 is sufficient for the ER membrane retention and that P450 is not recycled from the Golgi compartemnts to the ER in cells.
Isolation of yeast mutants defective in the dilysine motif-dependent E R retention : To identify the protein machinery for retention of dilysinetagged membrane proteins to the ER,we developed an assay to monitor ER retention of these proteins in yeast, making use of a fusion protein between invertase and a dilysine motif of Wbp1p. We isolated mutants with defective retention of invertase-Wbp1p to the ER by mutagenizing cells expressing invertase-Wbp1p and screening for mutants that transport the fusion protein to the plasma membrane by in situ invertase assay. 6 of the mutants exhibited a conditional growth phenotype with no growth at the restrictive temperature. One of the ts-mutants exhibited defect in protein transport from the ER to the Golgi aparatus.
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