Role of Human Sec63 in Modulating the Steady-State Levels of Multi-Spanning Membrane Proteins 英文参考文献.docVIP

RoleofHumanSec63inModulatingtheSteady-State LevelsofMulti-SpanningMembraneProteins AndreasMades1.,KatherinaGotthardt2.,KarinAwe2,JensStieler2,TatjanaDo¨ring2,SabineFu¨ser2, ReinhildPrange2* 1DepartmentofMedicineIII,HematologyandOncology,JohannesGutenberg-UniversitySchoolofMedicine,Mainz,Germany,2DepartmentofMedicalMicrobiology andHygiene,UniversityMedicalCenteroftheJohannesGutenbergUniversityMainz,Mainz,Germany Abstract TheSec61transloconoftheendoplasmicreticulum(ER)membraneformsanaqueouspore,allowingpolypeptidestobe transferredacrossorintegratedintomembranes.ProteintranslocationintotheERcanoccurco-andposttranslationally.In yeast, posttranslational translocation involves the heptameric translocase complex including its Sec62p and Sec63p subunits. The mammalian ER membrane contains orthologs of yeast Sec62p and Sec63p, but their function is poorly understood. Here, we analyzed the effects of excess and deficit Sec63 on various ER cargoes using human cell culture systems. The overexpression of Sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteinsinacotranslationalmode,whilesolubleandsingle-spanningERreportersarenotaffected.Consistentwiththis,the knock-down of Sec63 increases the steady-state pools of polytopic ER proteins, suggesting a substrate-specific and regulatory function of Sec63 in ER import. Overexpressed Sec63 exerts its down-regulating activity on polytopic protein levelsindependentofitsSec62-interactingmotif,indicatingthatitmaynotactinconjunctionwithSec62inhumancells. ThespecificactionofSec63isfurthersustainedbyourobservationsthattheup-regulationofeitherSec62ortwootherER proteins with lumenal J domains, like ERdj1 and ERdj4, does not compromise the steady-state level of a multi-spanning membranereporter.AJdomain-specificmutationofSec63,proposedtoweakenitsinteractionwiththeERresidentBiP chaperone, reduces the down-regulating capacity of excess Sec63, suggesting an involvement of BiP in this process. Together, these r