Alpha-COPI Coatomer Protein Is Required for Rough Endoplasmic Reticulum Whorl Formation in Mosquito Midgut Epithelial Cells 英文参考文献.docVIP

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Alpha-COPI Coatomer Protein Is Required for Rough Endoplasmic Reticulum Whorl Formation in Mosquito Midgut Epithelial Cells 英文参考文献.doc

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Alpha-COPI Coatomer Protein Is Required for Rough Endoplasmic Reticulum Whorl Formation in Mosquito Midgut Epithelial Cells 英文参考文献

Alpha-COPICoatomerProteinIsRequiredforRough EndoplasmicReticulumWhorlFormationinMosquito MidgutEpithelialCells GuoliZhou1.¤,JunIsoe1.,W.AntonyDay2,RogerL.Miesfeld1* 1DepartmentofChemistryandBiochemistry,TheUniversityofArizona,Tucson,Arizona,UnitedStatesofAmerica,2ArizonaResearchLabs,TheUniversityofArizona, Tucson,Arizona,UnitedStatesofAmerica Abstract Background:OneoftheearlyeventsinmidgutepithelialcellsofAedesaegyptimosquitoesisthedynamicreorganizationof rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previousstudiesshowingthatfeedingonanaminoacidmealinducesTORsignalinginAe.aegypti,weusedproteomicsand RNAitofunctionallyidentifymidgutepithelialcellproteinsthatcontributetoRERwhorlformation. Methodology/PrincipalFindings:AdultfemaleAe.aegyptimosquitoesweremaintainedonsugaralone(unfed),orfedan aminoacidmeal,andthenmidgutepithelialcellswereanalyzedbyelectronmicroscopyandproteinbiochemistry.Thesize andnumberofRERwhorlsinmidgutepithelialcellswerefoundtodecreasesignificantlyafterfeeding,andseveralKDEL- containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgutmicrosomalproteinsisolatedfromunfedandaminoacidfedmosquitoes,andofthe127proteinsidentified,8were chosenascandidatewhorlformingproteins.ThreecandidateproteinswereCOPIcoatomersubunits(alpha,beta,beta’),all ofwhichappearedtobepresentathigherlevelsinmicrosomalfractionsfromunfedmosquitoes.UsingRNAitoknockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly,whileadeficiencyinalpha-COPIhadnoeffectonearlytrypsinproteinsynthesisorsecretion3hrpostbloodmeal (PBM),expressionoflatephaseproteasesat24hrPBMwascompletelyblocked. Conclusions:alpha-COPIwasfoundtoberequiredfortheformationofRERwhorlsinmidgutepithelialcellsofunf