アクティブボード・２００８年１０月
　　　　　・・・・・２００８年１０月　６日更新・・・・・

研究発表を行った学会；
・VIIth International Symposium on Familial Amyloid Polyneuropathy and Ist International Workshop on Hereditary Amyloidosis.
　２００８年９月２日～５日(London, UK)

タイトル；The role of coat protein complex II machinery on the secretion, endoplasmic reticulum retention and endoplasmic reticulum-associated degradation of transthyretin.
　
発表者；　佐藤　卓志　氏
　　　（熊本大学　大学院薬学教育部　遺伝子機能応用学分野）
Abstract；
　The secretion of transthyretin (TTR) variants contributes to the pathogenesis of amyloidosis by forming aggregates in the extracellular environment. Recently, we demonstrated that unlike for wild-type TTR, the endoplasmic reticulum (ER) quality control system differentially regulates the fate of the TTR variants and their monomeric counterparts. However, the molecular mechanisms of secretion, ER retention and ER-associated degradation (ERAD) of TTR variants remain unknown. The coat protein complex II (COPII) machinery that is regulated by the small GTPase Sar1 is an important factor in the ER-to-Golgi trafficking of proteins. Therefore, in this study, we examined the contribution of COPII to the export of TTR to the Golgi and its potential contribution to the ER retention and ERAD pathway in mammalian cells. Secretion of wild-type and V30M TTRs were significantly inhibited by co-transfection with dominant negative mutant of Sar1, Sar1[H79G] referred to as Sar1-GTP, that inhibits hydrolysis of Sar1 and blocks COPII vesicle transport to the Golgi. Consistent with Sar1-GTP co-transfection experiments, secretion of wild-type and V30M TTRs were also markedly inhibited by treatment of the protein kinase inhibitor H89, which blocks recruitment of COPII coat components and prevents vesicle formation from the ER. These results demonstrate that COPII machinery is essential for export of the normally secreted TTRs such as wild-type and V30M TTRs from the ER. Moreover, immunofluorescence analyses of wild-type and V30M TTRs indicated that the intracellular localization of wild-type and V30M TTRs shifted from ER and Golgi compartments to Golgi compartment by treatment of the vacuolar H+-ATPase inhibitor bafilomycin A1, which inhibits retrograde transport from the Golgi to ER. These results suggest that the normally secreted TTRs may cycle between ER and Golgi. Next, we investigated the role of COPII machinery in the ER retention and ERAD of TTR variants using ER-retained TTR, D18G TTR. ERAD efficiency of D18G TTR did not change by co-transfection with Sar1-GTP or H89 treatment. Furthermore, D18G TTR did not co-localize with KDEL receptor and Sec23, a model of COPII cargo and a COPII coat component, respectively. These results suggest that ER retention of D18G TTR reflects the inability of D18G TTR to link to COPII vesicle, and COPII machinery is not required for ERAD of D18G TTR. Taken together, COPII machinery regulates the fate of wild-type TTR and TTR variants in the ER.