title: Multivesicular body formation requires OSBP-related proteins and cholesterol.
abstract: In eukaryotes, different subcellular organelles have distinct cholesterol concentrations, which is thought to be critical for biological functions. Oxysterol-binding protein-related proteins (ORPs) have been assumed to mediate nonvesicular cholesterol trafficking in cells; however, their in vivo functions and therefore the biological significance of cholesterol in each organelle are not fully understood. Here, by generating deletion mutants of ORPs in Caenorhabditis elegans, we show that ORPs are required for the formation and function of multivesicular bodies (MVBs). In an RNAi enhancer screen using obr quadruple mutants (obr-1; -2; -3; -4), we found that MVB-related genes show strong genetic interactions with the obr genes. 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 embryonic lethality upon knockdown of MVB-related genes. Finally, we show that knockdown of ORP1L, a mammalian ORP family member, induces the formation of enlarged MVBs in HeLa cells. Our in vivo findings suggest that the proper cholesterol level of late endosomes/lysosomes generated by ORPs is required for normal MVB formation and MVB-mediated membrane protein degradation.
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kobuna
authors.forename: Takao
authors.initials: T
authors.lastname: Inoue
authors.forename: Machiko
authors.initials: M
authors.lastname: Shibata
authors.forename: Keiko
authors.initials: K
authors.lastname: Gengyo-Ando
authors.forename: Akitsugu
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Shohei
authors.initials: S
authors.lastname: Mitani
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Arai
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D005810:Multigene Family; D057146:Multivesicular Bodies / Q000378:metabolism*; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
chemical_list: D029742:Caenorhabditis elegans Proteins; D011987:Receptors, Steroid; D000098825:Oxysterol Binding Proteins; D002784:Cholesterol