Showing papers by "Elina Ikonen published in 2011"

Role of ORPs in Sterol Transport from Plasma Membrane to ER and Lipid Droplets in Mammalian Cells

Abstract: In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.

Sterol binding by OSBP-related protein 1L regulates late endosome motility and function

Abstract: ORP1L is an oxysterol binding homologue that regulates late endosome (LE) positioning. We show that ORP1L binds several oxysterols and cholesterol, and characterize a mutant, ORP1L Δ560–563, defective in oxysterol binding. While wild-type ORP1L clusters LE, ORP1L Δ560–563 induces LE scattering, which is reversed by disruption of the endoplasmic reticulum (ER) targeting FFAT motif, suggesting that it is due to enhanced LE–ER interactions. Endosome motility is reduced upon overexpression of ORP1L. Both wild-type ORP1L and the Δ560–563 mutant induce the recruitment of both dynactin and kinesin-2 on LE. Most of the LE decorated by overexpressed ORP1L fail to accept endocytosed dextran or EGF, and the transfected cells display defective degradation of internalized EGF. ORP1L silencing in macrophage foam cells enhances endosome motility and results in inhibition of [3H]cholesterol efflux to apolipoprotein A-I. These data demonstrate that LE motility and functions in both protein and lipid transport are regulated by ORP1L.

Synthesis and Biosynthetic Trafficking of Membrane Lipids

Abstract: Eukaryotic cells can synthesize thousands of different lipid molecules that are incorporated into their membranes. This involves the activity of hundreds of enzymes with the task of creating lipid diversity. In addition, there are several, typically redundant, mechanisms to transport lipids from their site of synthesis to other cellular membranes. Biosynthetic lipid transport helps to ensure that each cellular compartment will have its characteristic lipid composition that supports the functions of the associated proteins. In this article, we provide an overview of the biosynthesis of the major lipid constituents of cell membranes, that is, glycerophospholipids, sphingolipids, and sterols, and discuss the mechanisms by which these newly synthesized lipids are delivered to their target membranes.

Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures

Abstract: Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR- and F-BAR-domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR-domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

Role for LAMP-2 in endosomal cholesterol transport

Abstract: The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein-2 (LAMP-2) and LAMP-1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP-1 and LAMP-2 (LAMP−/−), low-density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild-type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP-2 deficient liver and that overexpression of LAMP-2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP−/− cells was diminished by overexpression of any of the three isoforms of LAMP-2, but not by LAMP-1. The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.

Lipid droplet biogenesis: when the endoplasmic reticulum starts to fatten up.

Abstract: Lipid droplets are energy storage organelles composed of a phospholipid monolayer surrounding a hydrophobic core of neutral lipids, mainly triacylglycerols (TAGs) and steryl esters [1]. The exact mechanisms involved in the biogenesis of lipid droplets are not well understood, as direct imaging of newly forming lipid droplets is impossible with current methodology. Nevertheless, it is widely accepted that lipid droplets form within the endoplasmic reticulum (ER) membrane, in which neutral lipid synthesis and coalescence eventually lead to a droplet budding off the membrane, with the cytoplasmic leaflet of the ER membrane forming the monolayer of the lipid droplet [2,3]. Recent advances in understanding lipid droplet biogenesis have come from studies in yeast, using mutant strains to control specific steps in neutral lipid synthesis and lipid droplet formation.