Article pubs.acs.org/jpr
Change of Carbon Source Causes Dramatic Effects in the PhosphoProteome of the Archaeon Sulfolobus solfataricus D. Esser,† T. K. Pham,‡ J. Reimann,§ S. V. Albers,§ B. Siebers,*,† and P. C. Wright‡ †
Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Faculty of Chemistry, University of Duisburg-Essen, Universitätsstraße 5, 45141 Essen, Germany ‡ ChELSI Institute, Department of Chemical and Biological Engineering, The University of Sheffield, Mappin Street, Sheffield, S1 3JD, United Kingdom § Molecular Biology of Archaea, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse, 35043 Marburg, Germany S Supporting Information *
ABSTRACT: Protein phosphorylation is known to occur in Archaea. However, knowledge of phosphorylation in the third domain of life is rather scarce. Homology-based searches of archaeal genome sequences reveals the absence of two-component systems in crenarchaeal genomes but the presence of eukaryotic-like protein kinases and protein phosphatases. Here, the influence of the offered carbon source (glucose versus tryptone) on the phospho-proteome of Sulfolobus solfataricus P2 was studied by precursor acquisition independent from ion count (PAcIFIC). In comparison to previous phospho-proteome studies, a high number of phosphorylation sites (1318) located on 690 phospho-peptides from 540 unique phospho-proteins were detected, thus increasing the number of currently known archaeal phosphoproteins from 80 to 621. Furthermore, a 25.8/20.6/53.6 Ser/Thr/Tyr percentage ratio with an unexpectedly high predominance of tyrosine phosphorylation was detected. Phospho-proteins in most functional classes (21 out of 26 arCOGs) were identified, suggesting an important regulatory role in S. solfataricus. Focusing on the central carbohydrate metabolism in response to the offered carbon source, significant changes were observed. The observed complex phosphorylation pattern hints at an important physiological function of protein phosphorylation in control of the central carbohydrate metabolism, which might particularly operate in channeling carbon flux into the respective metabolic pathways. KEYWORDS: Archaea, Sulfolobus solfataricus, Phospho-proteomics, Protein phosphorylation, PAcIFIC, Central carbohydrate metabolism
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eukaryotic control points are absent,3e and only minor regulation has been observed at the transcript and proteome level in response to carbon source alteration.3c Reversible protein phosphorylation/dephosphorylation plays a major role in signal transduction and allows for a rapid appropriate cellular response to diverse external and internal signals/stimuli.5 This post-translational modification takes usually place at His and Asp residues (one-/two-component systems) or at Ser, Thr, or Tyr residues (eukaryotic-like protein kinases (ePKs)). Reversible protein phosphorylation was established in the Archaea with a report on the halophilic Euryarchaeon Halobacterium salinarium,6 where CheA and CheY represent the best-studied archaeal two-component system. Archaeal phospho (p)-proteins with Ser, Thr, and Tyr modifications have since been identified in the thermoacidophilic Crenarchaeon S. acidocaldarius.7 It is also clear that eukaryotic
INTRODUCTION
As well as constituting the third domain of life, Archaea offer exciting opportunities for biotechnological exploitation. The key model organism for the Crenarchaeota is Sulfolobus solfataricus, a thermoacidophile that grows optimally at 80 °C and pH 3.2. 1 It grows heterotrophically on complex peptidolytic constituents and a wide variety of sugars.2 The central carbohydrate metabolism (CCM) has been extensively studied,3 and due to new, unusual enzymes, pathways, and regulatory potential it shows great potential for White Biotechnology and Synthetic Biology.4 S. solfataricus uses non-phosphorylative modifications of the respective hexose and pentose degradation pathways, like the branched Entner−Doudoroff (ED) pathway for glucose and galactose,3a,e,f the Dahms pathway for D-xylose degradation,3b and an oxidative pathway for D-arabionse degradation.3d Like most other Archaea, S. solfataricus uses the modified Embden− Meyerhof−Parnas (EMP) pathway for gluconeogenesis.3c,e Despite these metabolic pathway investigations, their regulation is still unclear. It is known though that classical bacterial and © 2012 American Chemical Society
Received: February 28, 2012 Published: May 29, 2012 4823
dx.doi.org/10.1021/pr300190k | J. Proteome Res. 2012, 11, 4823−4833
Journal of Proteome Research
Article
RC-DC Protein Quantification Assay (Bio-Rad, UK). All chemicals were purchased from Sigma.
protein Ser/Thr and Tyr kinases (ePK) and the respective protein phosphatases (PP) are present in Archaea.8 Further, bioinformatic analyses revealed the presence of ePKs and PPs in all completed archaeal genomes, whereas His kinases twocomponent systems are restricted to Euryarchaeota.5,9 It is also proposed that novel PK families exist among the Archaea.10 In terms of phosphorylation-mediated post-translational regulation of the CCM, only for gluconate dehydratase (GAD) has this unambiguously been demonstrated,11 raising the question as to whether post-translational modifications such as phosphorylation plays a major role in CCM regulation in S. solfataricus and the Archaea in general. The largest archaeal p-proteome study to date, the pproteome from H. salinarum strain R1 was analyzed using TiO2 enrichment of p-peptides, and mass spectrometry revealed 69 broadly functionally distributed p-proteins.12 Despite this background, knowledge on post-translational modifications in Archaea is rather scarce. Only five PKs and six PPs have been characterized in detail and only for one of these regulatory function has been demonstrated.5,13 We demonstrate that the precursor acquisition independent from ion count (PAcIFIC) approach14 can be extended to deeply mine p-proteomes. We exemplify this by studying a member of the third domain of life. Importantly, using PAcIFIC, compared to previous studies depending on ppeptide pre-enrichment approaches we detected a 3−7-fold higher number of p-proteins with a so far unique/unexpected preference for Tyr-P (1318 phosphorylation sites located on 690 p-peptides from 540 p-proteins with a Ser/Thr/Tyr-ratio of 25.8/20.6/53.6%). Using the pre-enrichment free PACiFIC approach, we dramatically extend knowledge of archaeal pproteins from 80 to 620. Taking advantage of our workflow, we demonstrate the substantial impact of offered carbon source on phosphorylation events occurring in S. solfataricus, in particular, those in the CCM. This sets the scene for future deeper functional studies to assess exactly the underlying processes.
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Protein Digestion and Fractionation
Two milligrams of protein (per condition) were dissolved and denatured in 8 M urea, reduced with 10 mM DTT in 50 mM ammonium bicarbonate at 56 °C for 1 h, then alkylated with 55 mM idoacetamide in 50 mM ammonium bicarbonate at 37 °C for 30 min in the dark. Samples were then diluted with 40 mM ammonium bicarbonate in 9% (v/v) acetonitrile (ACN) to reach a final
