<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Proximity biotinylation at organelle contact sites reveals GRAMD1B as a sterol transporter acting at the lysosome-ER interface"^^ . "Contact sites between membranes of different cellular organelles are involved in a wide variety of biological processes. While many of the key factors involved in their formation have been identified and many proteins that act primarily at these contact sites have been studied extensively, a comprehensive picture of both the architecture but also the function of these contacts is still missing. This lack of knowledge is to a large degree due to the small dimensions of these interfacing membranes, which make their identification and analysis using conventional methods rather challenging. This work establishes a proximity-labelling assay based on a split biotin ligase connected to known protein tether-pairs at three distinct contact sites of the endoplasmic reticulum (ER) with lysosomes, mitochondria and endosomes, respectively. Proteins that were biotinylated at these membrane contact sites were then identified by mass spectrometry based proteomics. The resulting list of identified hits contained both novel proteins, as well as proteins that had previously been reported to fulfill a role at membrane contact sites, highlighting the efficacy of the assay.\r\nThe function of one such protein, GRAMD1B, was investigated further. This protein is essential for non-vesicular cholesterol import to the ER from the plasma membrane, but was also found to locate to contact sites with lysosomes and interact with the major lysosomal cholesterol exporting protein, NPC1. Results of the split biotin ligase assay confirmed a localization at lysosome-ER contact site, which led us to further study its function at this contact. Using a lysosomally prelocalized, modified cholesterol probe in GRAMD1B-overexpressing cells, pulse-chase measurements of cholesterol esterification suggested an involvement of GRAMD1B in two separate lysosome-to-ER transport processes with different temporal scales: A fast and direct cholesterol transport at lysosome-ER contacts dependent on NPC1 as well as an import from the plasma membrane to the ER at slower timescales. This was substantiated by the fact that the impact of GRAMD1B-overexpression on the faster transport process was reduced by NPC1-deficiency, while trapping of cholesterol at the plasma membrane only reduced the effect at later time points. In addition, live-cell microscopic analysis showed GRAMD1B’s ability to recruit ER tubules to cholesterol-laden lysosomes and confirmed a dependency on the presence of NPC1 in the lysosomal membrane. This further strengthens the hypothesis of a dual role of GRAMD1B in the transport of cholesterol to the ER, with spatial coupling of two cholesterol transporters at the lysosome-ER interface. Overall, this work shows that proximity labelling using split biotin ligase fragments fused to known contact site tether pairs is a powerful tool to identify proteins acting at organelle contacts. One such protein, GRAMD1B, acts in multiple routes of subcellular cholesterol trafficking, where this work focusses on its function in direct lysosome-to-ER transport of sterols. The applied methods, particularly the use of modified cholesterol probes prelocalized to lysosomes to observe lysosomal egress, as well as the insights gained from studying GRAMD1B’s actions at lysosome-ER contacts, will contribute to a more comprehensive understanding of subcellular cholesterol homeostasis, which, in turn, is relevant for a wide variety of physiological and pathophysiological processes."^^ . "2022" . . . . . . . "Valentin"^^ . "Schoop"^^ . "Valentin Schoop"^^ . . . . . . "Proximity biotinylation at organelle contact sites reveals GRAMD1B as a sterol transporter acting at the lysosome-ER interface (PDF)"^^ . . . "Valentin-Schoop_Dissertation.pdf"^^ . . . "Proximity biotinylation at organelle contact sites reveals GRAMD1B as a sterol transporter acting at the lysosome-ER interface (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Proximity biotinylation at organelle contact sites reveals GRAMD1B as a sterol transporter acting at the lysosome-ER interface (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Proximity biotinylation at organelle contact sites reveals GRAMD1B as a sterol transporter acting at the lysosome-ER interface (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Proximity biotinylation at organelle contact sites reveals GRAMD1B as a sterol transporter acting at the lysosome-ER interface (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Proximity biotinylation at organelle contact sites reveals GRAMD1B as a sterol transporter acting at the lysosome-ER interface (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #31765 \n\nProximity biotinylation at organelle contact sites reveals GRAMD1B as a sterol transporter acting at the lysosome-ER interface\n\n" . "text/html" . . . "500 Naturwissenschaften und Mathematik"@de . "500 Natural sciences and mathematics"@en . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .