HEK293 Flp-In T-Rex were authenticated by STR analysis with The Center for Applied Genomics Genetic Analysis Facility (Sick Kids Hospital, Toronto). HeLa cells and primary fibroblasts were not independently authenticated Mycoplasma contaminationCell lines were routinely monitored for mycoplasma contamination as assessed by a commercial kit (MycoAlert, Lonza). Commonly misidentified lines (See ICLAC register)No commonly misidentified cell lines were used in this study.

INTRODUCTIONCompartmentalization is an essential characteristic of eukaryotic cells, ensuring that cellular processes are partitioned to defined subcellular locations. High throughput microscopy 1 and biochemical fractionation coupled with mass spectrometry 2-6 have helped to define the proteomes of multiple organelles and macromolecular structures. However, many compartments have remained refractory to such methods, partly due to lysis and purification artefacts and poor subcompartment resolution. Recently developed proximity-dependent biotinylation approaches such as BioID and APEX provide an alternative avenue for defining the composition of cellular compartments in living cells (e.g. 7-10 ). Here we report an extensive BioID-based proximity map of a human cell, comprising 192 markers from 32 different compartments that identifies 35,902 unique high confidence proximity interactions and localizes 4,145 proteins expressed in HEK293 cells. The recall of our localization predictions is on par with or better than previous large-scale mass spectrometry and microscopy approaches, but with higher localization specificity. In addition to assigning compartment and subcompartment localization for many previously unlocalized proteins, our data contain finegrained localization information that, for example, allowed us to identify proteins with novel roles in mitochondrial dynamics. As a community resource, we have created humancellmap.org, a website that allows exploration of our data in detail, and aids with the analysis of BioID experiments. BODYProximity-dependent labelling approaches have rapidly grown in popularity, as they provide a robust way to label the environment in which a protein resides in living cells 7,8 . In the most widely used of these techniques, BioID, a mutant E. coli biotin ligase -BirA* (R118G) -is fused in-frame with the coding sequence of a bait polypeptide of interest, and the resulting fusion protein expressed in cultured cells. While BirA* can activate biotin to biotinoyl-AMP, the abortive mutant enzyme exhibits a reduced affinity for the activated molecule. A reactive intermediate is thus released into the local environment that can react with free epsilon amine groups on nearby lysine residues 7 . This ability for BirA* to label a local environment has led to BioID being employed by multiple laboratories to define the composition, and in some cases the overall organization, of both membrane-bound and membraneless organelles (e.g. 7-10 ).Here, we set out to map a human cell by profiling markers (consisting of full-length proteins or targeting sequences) from 32 cellular compartments. These compartments include the cytosolic face of all membrane-bound organelles, the ER lumen, subcompartments of the nucleus and mitochondria, major membraneless organelles such as the centrosome and the nucleolus, and the main cytoskeletal structures (actin, microtubules and intermediate filaments). Several proteins were also queried throughout the endomembrane system to identify components enriched at locales a...

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.