RESUMO
Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.
Assuntos
Colesterol/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Multimerização Proteica , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico , Sistemas CRISPR-Cas/genética , LDL-Colesterol/metabolismo , Endossomos/metabolismo , Endossomos/ultraestrutura , Corantes Fluorescentes/metabolismo , Genoma Humano , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Hidroximetilglutaril-CoA Sintase/metabolismo , Lisossomos/ultraestrutura , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Proteína C1 de Niemann-Pick , Ligação Proteica , proteínas de unión al GTP Rab7RESUMO
HIV-1 encodes four "accessory proteins" (Vif, Vpr, Vpu, and Nef), dispensable for viral replication in vitro but essential for viral pathogenesis in vivo. Well characterized cellular targets have been associated with Vif, Vpu, and Nef, which counteract host restriction and promote viral replication. Conversely, although several substrates of Vpr have been described, their biological significance remains unclear. Here, we use complementary unbiased mass spectrometry-based approaches to demonstrate that Vpr is both necessary and sufficient for the DCAF1/DDB1/CUL4 E3 ubiquitin ligase-mediated degradation of at least 38 cellular proteins, causing systems-level changes to the cellular proteome. We therefore propose that promiscuous targeting of multiple host factors underpins complex Vpr-dependent cellular phenotypes and validate this in the case of G2/M cell cycle arrest. Our model explains how Vpr modulates so many cell biological processes and why the functional consequences of previously described Vpr targets, identified and studied in isolation, have proved elusive.
