ABSTRACT
Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.
Subject(s)
Protein Interaction Mapping , Proteins/metabolism , Proteome/metabolism , Proteomics/methods , CRISPR-Cas Systems , Cluster Analysis , Datasets as Topic , Fluorescent Dyes , HEK293 Cells , Humans , Immunoprecipitation , Machine Learning , Mass Spectrometry , Microscopy, Confocal , RNA-Binding Proteins/metabolism , Spatial AnalysisABSTRACT
Signaling pathways that involve diatomic gases in photosynthetic organisms are not well understood. Exposure to nitric oxide or carbon monoxide is known to elicit certain responses in some photosynthetic organisms. For example, Chlamydomonas reinhardtii grown in low-iron media responds to exogenous carbon monoxide by increasing cell growth and intracellular chlorophyll levels. Here, we characterize Cyg11, a gas-responsive soluble guanylate cyclase from the eukaryotic green alga C. reinhardtii that converts GTP to cGMP. Cyg11 transcription is upregulated when C. reinhardtii is grown in iron-limited media, suggesting its importance in nutrient-limited environments. Cyg11 is purified as a homodimer and is activated by nitric oxide (2.5-fold over basal activity) and carbon monoxide (6.3-fold). The heme binding stoichiometry of Cyg11 was found to be one heme per homodimer, an unexpected result based on the sequence and oligomerization state of the enzyme. Gas binding properties, the kinetics of gas binding, and the ligand-modulated activity of Cyg11 are consistent with CO as the relevant physiological ligand.
