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.
Highlights
The multivesicular body (MVB) sorting pathway provides a mechanism for the lysosomal degradation of membrane proteins and has a role in many processes, including growth factor receptor down-regulation [1], antigen presentation [2], developmental signaling [3,4], the budding of enveloped viruses [5], and cytokinesis [6,7]
We found that disruption of oxysterol-binding protein (OSBP)–related proteins (ORPs), which have been proposed to function in cellular cholesterol distribution and metabolism, reduces the cholesterol content in late endosomes/lysosomes, leading to impaired MVB function
MVB sorting pathway is known to be involved in many processes, including growth factor receptor down-regulation, exosome secretion, antigen presentation, the budding of enveloped viruses, and cytokinesis
Summary
The multivesicular body (MVB) sorting pathway provides a mechanism for the lysosomal degradation of membrane proteins and has a role in many processes, including growth factor receptor down-regulation [1], antigen presentation [2], developmental signaling [3,4], the budding of enveloped viruses [5], and cytokinesis [6,7]. MVBs form when the limiting membrane of the late endosomes invaginates and buds into the lumen of the organelle, selecting a subset of the proteins from the limiting membrane in the process [8,9]. The MVB sorting machinery is constituted by proteins that form the endosomal sorting complexes required for transport (ESCRT-I, -II, and -III) [10,11]. These ESCRT complexes are recruited sequentially to endosomal membranes where they function in sorting cargo and generating characteristic intralumenal vesicles. In addition to the ESCRT proteins, lipid molecules have been assumed to be involved in MVB formation by creating local microdomains in the endosomal membrane that induce the inward membrane curvature. Treatment with anti-LBPA antibodies disrupts normal MVB formation in mammalian cells, suggesting that LBPA has a role in driving lumenal-vesicle formation at the cellular level [14]
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