A proximity-dependent biotinylation map of a human cell: an interactive web resource

Abstract

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...

Dual Proteome-scale Networks Reveal Cell-specific Remodeling of the Human Interactome

“…Proximity labeling could capture weaker, more transient interactions and aid study of multi-pass membrane proteins. Indeed, large-scale proximity labeling is underway to define proteome spatial organization (Go et al, 2019), providing a complementary view of protein interactions.…”

Nucleolar Organization and Functions in Health and Disease

“…Correll and colleagues present a comprehensive review of the structure and function of the human nucleolus [1]. Integrating classic ultrastructural and biochemical evidence with recent advances from modern biophysical techniques and cryoEM, these authors describe a compartment that remains intact despite the continuous exchange of components and rapid flux of ribosome intermediates.…”

“…Recent studies in vitro and in vivo provided new insights that further substantiate the importance of nucleoli for senescence and aging at the cellular or organismal level. These studies are being discussed in several reviews published in Cells, including this Special Issue and previous publications [1,9,22,25].…”

“…Corell et al [1] point out an interesting conundrum, related to the nucleolus in aging cells. In HGPS patient cells rDNA transcription is elevated.…”

Multiplexed Proximity Biotinylation Coupled to Mass Spectrometry for Defining Integrin Adhesion Complexes

“…Nevertheless, BioID has proven effective for examining proximal interactors of individual proteins and larger complexes that are typically difficult to study, such as cell-cell contacts, the centrosome, and IACs (Chastney et al, 2020;Dong et al, 2016;Gupta et al, 2015;Van Itallie et al, 2013). In larger-scale network mapping studies, multiplexed BioID has been used to reveal the spatial relationships between components of large complexes, and even between multiple organelles (Chastney et al, 2020;Go et al, 2019;Gupta et al, 2015;Youn et al, 2018). In such initiatives, careful analysis is required to interpret the data and to infer potential biological insight.…”