Multivesicular Body Formation Requires OSBP–Related Proteins and Cholesterol 英文参考文献.docVIP

MultivesicularBodyFormationRequiresOSBP–Related ProteinsandCholesterol HiroyukiKobuna1,TakaoInoue1,2*.,MachikoShibata1,KeikoGengyo-Ando2,3¤,AkitsuguYamamoto4, ShoheiMitani2,3,HiroyukiArai 1,2 . * 1GraduateSchoolofPharmaceuticalSciences,UniversityofTokyo,Tokyo,Japan,2CoreResearchforEvolutionalScienceandTechnology(CREST),JapanScienceand TechnologyAgency(JST),Tokyo,Japan,3DepartmentofPhysiology,TokyoWomen’sMedicalUniversitySchoolofMedicine,Tokyo,Japan,4DepartmentofBio-Science, NagahamaInstituteofBio-ScienceandTechnology,Nagahama,Japan Abstract Ineukaryotes,differentsubcellularorganelleshavedistinctcholesterolconcentrations,whichisthoughttobecriticalfor biological functions. Oxysterol-binding protein-related proteins (ORPs) have been assumed to mediate nonvesicular cholesteroltraffickingincells;however,theirinvivofunctionsandthereforethebiologicalsignificanceofcholesterolineach organellearenotfullyunderstood.Here,bygeneratingdeletionmutantsofORPsinCaenorhabditiselegans,weshowthat ORPsarerequiredfortheformationandfunctionofmultivesicularbodies(MVBs).InanRNAienhancerscreenusingobr quadruplemutants(obr-1;-2;-3;-4),wefoundthatMVB–relatedgenesshowstronggeneticinteractionswiththeobrgenes. In obr quadruple mutants, late endosomes/lysosomes are enlarged and membrane protein degradation is retarded, although endocytosed soluble proteins are normally delivered to lysosomes and degraded. We also found that the cholesterol content of late endosomes/lysosomes is reduced in the mutants. In wild-type worms, cholesterol restriction induces the formation of enlarged late endosomes/lysosomes, as observed in obr quadruple mutants, and increases embryoniclethalityuponknockdownofMVB–relatedgenes.Finally,weshowthatknockdownofORP1L,amammalianORP family member, induces the formation of enlarged MVBs in HeLa cells. Our in vivo findings suggest that the proper cholesterolleveloflateendosomes/lysosomesgeneratedbyORPsisrequiredfornormalMVBformationandMVB–mediated membraneproteindegradation. Cit