ABSTRACT
Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of cross-links improve the sensitivity and accuracy of correct cross-link identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multiplatform.
Subject(s)
Proteomics , Tandem Mass Spectrometry , Proteomics/methods , Tandem Mass Spectrometry/methods , Peptides/analysis , Proteins/chemistry , Software , Cross-Linking Reagents/chemistryABSTRACT
Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies, which associate helically to template MT growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC.
Subject(s)
Antigens, Nuclear/genetics , Microtubules/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Tubulin/chemistry , Tubulin/genetics , Binding Sites , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Cryoelectron Microscopy/methods , Crystallography, X-Ray/methods , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Humans , Mass Spectrometry/methods , Microtubule-Organizing Center , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Tubulin/classification , Tubulin/metabolismABSTRACT
Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.
Subject(s)
Macromolecular Substances , Multiprotein Complexes/physiology , Animals , Biochemical Phenomena , Cell Physiological Phenomena , Cytoplasm/chemistry , Cytoplasm/genetics , Cytoplasm/metabolism , Eukaryotic Cells/chemistry , Eukaryotic Cells/metabolism , Eukaryotic Cells/physiology , Humans , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Multiprotein Complexes/chemistry , Organelles/chemistry , Organelles/genetics , Organelles/metabolism , Protein Aggregates/physiologyABSTRACT
The microtubule (MT) cytoskeleton plays important roles in many cellular processes. In vivo, MT nucleation is controlled by the γ-tubulin ring complex (γTuRC), a 2.1-MDa complex composed of γ-tubulin small complex (γTuSC) subunits. The mechanisms underlying the assembly of γTuRC are largely unknown. In yeast, the conserved protein Spc110p both stimulates the assembly of the γTuRC and anchors the γTuRC to the spindle pole body. Using a quantitative in vitro FRET assay, we show that γTuRC assembly is critically dependent on the oligomerization state of Spc110p, with higher-order oligomers dramatically enhancing the stability of assembled γTuRCs. Our in vitro findings were confirmed with a novel in vivo γTuSC recruitment assay. We conclude that precise spatial control over MT nucleation is achieved by coupling localization and higher-order oligomerization of the receptor for γTuRC.
