Proteome Turnover in the Spotlight: Approaches, Applications, and Perspectives.

In all cells, proteins are continuously synthesized and degraded to maintain protein homeostasis and modify gene expression levels in response to stimuli. Collectively, the processes of protein synthesis and degradation are referred to as protein turnover. At a steady state, protein turnover is constant to maintain protein homeostasis, but in dynamic responses, proteins change their rates of synthesis and degradation to adjust their proteomes to internal or external stimuli. Thus, probing the kinetics and dynamics of protein turnover lends insight into how cells regulate essential processes such as growth, differentiation, and stress response. Here, we outline historical and current approaches to measuring the kinetics of protein turnover on a proteome-wide scale in both steady-state and dynamic systems, with an emphasis on metabolic tracing using stable isotope-labeled amino acids. We highlight important considerations for designing proteome turnover experiments, key biological findings regarding the conserved principles of proteome turnover regulation, and future perspectives for both technological and biological investigation.

The Xenobiotic Extrusion Mechanism of the MATE Transporter NorM_PS from Pseudomonas stutzeri.

Multidrug resistance (MDR) in bacterial pathogens has become a severe threat to public health. Membrane transporters of the multidrug and toxic compound extrusion (MATE) family contribute critically to MDR, making them promising drug targets. Despite recent advances, structures in different conformations and the mechanistic details of their antiport cycle are still elusive. Here we studied NorM_PS, a representative MATE transporter from Pseudomonas stutzeri, using biochemical assays in combination with hydrogen/deuterium exchange-mass spectrometry. Our results confirm that the antiport is proton dependent and electroneutral with a stoichiometry of two protons per one doubly positively charged substrate. We investigated the conformational dynamics upon substrate binding, and our hydrogen/deuterium exchange-mass spectrometry analysis revealed an occlusion in the proposed binding site as well as a closure of the cytoplasmic cavity and formation of a periplasmic cavity. Together with the results of selected variants (D38N, D373N and Q376A), we propose a six-step rocker-switch model for NorM_PS, which also increases our understanding of related MATE transporters and may help to fight the burden of MDR.

Ligand-induced conformational dynamics of the Escherichia coli Na+/H+ antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry.

Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of Escherichia coli has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li+ binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases.

Three multihaem cytochromes c from the hyperthermophilic archaeon Ignicoccus hospitalis: purification, properties and localization.

Three different multihaem cytochromes c were purified from cell extracts of the hyperthermophilic archaeon Ignicoccus hospitalis. One tetrahaem cytochrome, locus tag designation Igni_0530, was purified from membrane fractions together with the iron-sulfur protein Igni_0529. Two octahaem cytochromes, Igni_0955 and Igni_1359, were purified from soluble fractions but were also present in the membrane fraction. N-terminal sequencing showed that three of the four proteins had their signal peptides cleaved off, while results were ambiguous for Igni_0955. In contrast, mass spectrometry of Igni_0955 and Igni_1359 resulted in single mass peaks including the signal sequences and eight haems per subunit and so both forms might be present in the cell. Igni_0955 and Igni_1359 belong to the hydroxylamine dehydrogenase (HAO) family (29 % mutual identity). HAO or reductase activities with inorganic sulfur compounds were not detected. Igni_0955 was reduced by enriched I. hospitalis hydrogenase at a specific activity of 243 nmol min(-1) (mg hydrogenase)(-1) while activity was non-existent for Igni_0530 and low for Igni_1359. Immuno-electron microscopy of ultra-thin sections showed that Igni_0955 and Igni_1359 are located in both I. hospitalis membranes and also in the intermembrane compartment. We concluded that these cytochromes might function as electron shuttles between the hydrogenase in the outer cellular membrane and cellular reductases, whereas Igni_0530 might be part of the sulfur-reducing mechanism.