1995 Fiscal Year Final Research Report Summary
Membrane Dynamics and Its Regulation During Intracellular Vesicular Transport
| Project/Area Number |
05454643
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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 | The University of Tokyo |
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Principal Investigator |
NAKANO Akihiko The University of Tokyo, Faculty of Science Associate Professor, 大学院理学系研究科, 助教授 (90142140)
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| Project Period (FY) |
1993 – 1995
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| Keywords | protein secretion / endoplasmic reticulum / Golgi apparatus / vesicular transport / vesicle recycling / Sec12p / RER1 / Yeast (S.cerevisiae) |
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| Research Abstract |
Yeast Sec12p, a type II transmembrane glycoprotein, is required for formation of transport vesicles from the endoplasmic reticulum (ER). Biochemical and morphological analyzes have suggested that Sec12p is localized to the ER by two mechanisms : static retention in the ER and dynamic retrieval from the Golgi apparatus. The rer1 mutant we isolated mislocalizes the authentic Sec12p to the later compartments of the Golgi. To understand the role of RER1 on Sec12p localization, we cloned the gene. RER1 encodes a hydrophobic protein of 188 amino acid residues with four membrane spans. The rer1 null mutant is viable. Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER,implying that the retention system is still operating in the mutant. To determine the subcellular localization of Rer1p, an HA-epitope-tagged Rer1p were observed by immunofluorescence microscopy. The anti-HA monoclonal antibody stains the cells in a punctate pattern which is typical for Golgi proteins and clearly distinct from the ER staining. Subcellular fractionation experiments indicated that the Rer1p behaves like an early Golgi protein. From these, we conclude that Rer1p functions in the Golgi membrane to return Sec12p that has escaped from the static retention system of the ER.We have also performed a systematic analysis to determine the ER localization signals in Sec12p corresponding to these two mechanisms. Both the transmembrane domain (TMD) and the N-terminal domain of Sec12p show the ability to localize the protein to the ER.The effect of the TMD is potent and sufficient by itself for the ER localization and is strongly dependent on RER1. On the other hand, the N-terminal domain shows a moderate ER-localization capability which is independent of RER1. The rate of mannosyl modification has been measured to distinguish between retention and retrieval. The results suggest that the TMD mainly acts as the retrieval signal and the N-terminal domain the retention signal.
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Research Products
(20 results)
