Natalia, V. | Search bioRxiv

bioRxiv2026

Site-resolved spatial and structural interactome of a human cell

The spatial and structural arrangement of proteins determine virtually every process in human cells. We combined gentle subcellular fractionation by differential ultracentrifugation with cross-linking mass spectrometry to systematically map this cellular proteome architecture with residue-level evidence, identifying 164,146 residue-to-residue links in HEK293 cells. These links capture spatial protein arrangement at a resolution sufficient to pinpoint protein localizations at sub-organelle level, determine protein orientations within cellular membranes, and identify inter-organelle contact sites. The residue-level information provides evidence for 18,074 direct protein-protein interactions (PPIs), which we integrate into AlphaFold-based pipelines to nominate PPI-mediating short linear motifs and generate assembly models of large protein complexes. Guided by these spatial and structural readouts, we discover new PPIs within the endomembrane system that regulate the compartmental localization of trafficking machinery. Leveraging a network topology-driven strategy, we augment our HEK293 dataset with PPI data from different cell lines and methods, expanding the spatial and structural interactome of human cells.

systems biology↗

bioRxiv2023

Multi-functional ARF1 compartments serve as a hub for short-range cargo transfer to endosomes

Cellular membrane homeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range transport between the Golgi, endosomes and plasma membrane. Here we revisit the intracellular organization of the vesicular transport machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast-confocal) or spatial (stimulated emission depletion (STED)) microscopy as well as correlative light and electron microscopy. We characterize novel tubulo-vesicular ARF1 compartments that harbor clathrin and different APs. Our findings reveal two functionally different classes of ARF1 compartments, each decorated by a different combination of APs. Perinuclear ARF1 compartments facilitate Golgi export of secretory cargo, while peripheral ARF1 compartments are involved in endocytic recycling downstream of early endosomes. Contrary to the classical model of long-range vesicle shuttling, we observe that ARF1 compartments shed ARF1 and mature into recycling endosomes. This maturation process is impaired in the absence of AP-1 and results in trafficking defects. Collectively, these data highlight a crucial role for ARF1 compartments in post-Golgi sorting.

cell biology↗