DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift

Abstract

The yeastSaccharomyces cerevisiaeis a powerful model system for systems-wide biology screens and large-scale proteomics methods. Nearly complete proteomics coverage has been achieved owing to advances in mass spectrometry. However, it remains challenging to scale this technology for rapid and high-throughput analysis of the yeast proteome to investigate biological pathways on a global scale. Here we describe a systems biology workflow employing plate-based sample preparation and rapid, single-run, data-independent mass spectrometry analysis (DIA). Our approach is straightforward, easy to implement, and enables quantitative profiling and comparisons of hundreds of nearly complete yeast proteomes in only a few days. We evaluate its capability by characterizing changes in the yeast proteome in response to environmental perturbations, identifying distinct responses to each of them and providing a comprehensive resource of these responses. Apart from rapidly recapitulating previously observed responses, we characterized carbon source-dependent regulation of the GID E3 ligase, an important regulator of cellular metabolism during the switch between gluconeogenic and glycolytic growth conditions. This unveiled regulatory targets of the GID ligase during a metabolic switch. Our comprehensive yeast system readout pinpointed effects of a single deletion or point mutation in the GID complex on the global proteome, allowing the identification and validation of targets of the GID E3 ligase. Moreover, this approach allowed the identification of targets from multiple cellular pathways that display distinct patterns of regulation. Although developed in yeast, rapid whole-proteome–based readouts can serve as comprehensive systems-level assays in all cellular systems.

Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase

“…In total, 31 proteins were stabilized in the gid2 Δ, gid9 Δ, or ubc8 Δ mutants ( Figure 5 A). These included several GID subunits and three potential GID substrates (Aro10, Tma10, and Stf2) identified in a recent proteomic study ( Karayel et al., 2020 ). For most tFT fusions, their stabilization was detectable in colonies, which consist of cells in different metabolic states ( Cáp et al., 2012 ), but not in logarithmically growing cultures with glucose as a carbon source ( Figure 5 B).…”

GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

“…One speculative possibility could be that any potential advantage of avid binding is offset by accessibility of numerous ubiquitylation sites to GID SR4 . Future studies will be required to understand how Pck1 and other GID E3 substrates, including the Gid4 substrate receptor itself, are recognized and ubiquitylated ( Hämmerle et al., 1998 ; Karayel et al., 2020 ; Menssen et al., 2018 ).…”

“…Spectronaut version 13 (Biognosys) was used to analyze DIA raw files using the yeast FASTA file (Swissprot, 2018) and the proteome library previously published ( Karayel et al., 2020 ) with default settings and enabled cross run normalization. The Perseus software package version 1.6.0.7 was used for the data analysis ( Tyanova et al., 2016 ).…”

“…Much like well-studied multiprotein E3 ligases, such as anaphase-promoting complex/cyclosome (APC/C) or cullin-RING ligases, GID is not a singular complex—a core catalytic and scaffolding assembly is modulated by other subunits ( Barford, 2020 ; Karayel et al., 2020 ; Liu and Pfirrmann, 2019 ; Melnykov et al., 2019 ; Qiao et al., 2020 ; Rusnac and Zheng, 2020 ; Watson et al., 2019 ). The constituents of various GID assemblies and how they achieve regulation are beginning to emerge.…”

“…Coexpression of these subunits in insect cells enables purification of recombinant GID Ant and systematic interrogation of GID functions. Within the GID Ant scaffold, Gid5 can bind the structurally homologous, interchangeable substrate-binding receptors Gid4 and Gid10 ( Karayel et al., 2020 ; Melnykov et al., 2019 ; Qiao et al., 2020 ). Of these, the molecular basis of substrate binding by Gid4 is well understood: glucose-induced incorporation of Gid4 into the GID E3 enables recognition of substrate “Pro/N-degron” motifs depending on an N-terminal proline ( Chen et al., 2017 ; Dong et al., 2018 ; Hämmerle et al., 1998 ; Regelmann et al., 2003 ; Santt et al., 2008 ).…”