Abstract

BackgroundCellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes–associated protein whose expression level affects cellular free cholesterol content.Methodology/Principal FindingsUsing a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY.Conclusions/SignificanceLDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.

Highlights

  • Cholesterol is essential for maintenance of membrane integrity and multiple cellular functions

  • Internalized lipoprotein-associated cholesterol esters are hydrolyzed to free cholesterol in late endosome/lysosome (LE/ LY), from which it is exported to various destinations, including the plasma membrane and the endoplasmic reticulum

  • We propose that the regulatory function of Pdro on cellular cholesterol homeostasis implicates late endosome/lysosome (LE/LY) motility that is involved in low-density lipoproteins (LDL)-derived cholesterol traffic

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Summary

Introduction

Cholesterol is essential for maintenance of membrane integrity and multiple cellular functions. Internalized lipoprotein-associated cholesterol esters are hydrolyzed to free cholesterol in late endosome/lysosome (LE/ LY), from which it is exported to various destinations, including the plasma membrane and the endoplasmic reticulum. NPC disease is caused by mutations in NPC-1 and -2 proteins located in LE/LY that are believe to coordinate cholesterol egress from LE/LY, but the precise defect remains unknown. In addition to a role for NPC proteins, an underlying cause for cholesterol trafficking defects in NPC may be changes in the activity of proteins that regulate endosomal motility. Further description of the protein and lipid factors that control intracellular cholesterol transport and content are important for a better understanding of cholesterol homeostasis. We describe a late endosome/lysosomes–associated protein whose expression level affects cellular free cholesterol content

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