title: Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation.
abstract: Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Wan-Ling
authors.initials: WL
authors.lastname: Wu
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.|Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Shu-Jung
authors.initials: SJ
authors.lastname: Lai
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.|Ph.D program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung 40227, Taiwan.
authors.forename: Jhih-Tian
authors.initials: JT
authors.lastname: Yang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.|Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan.|Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Jeffy
authors.initials: J
authors.lastname: Chern
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.|Core Facilities for Protein Structural Analysis, Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Suh-Yuen
authors.initials: SY
authors.lastname: Liang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.|Core Facilities for Protein Structural Analysis, Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Chi-Chi
authors.initials: CC
authors.lastname: Chou
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Chih-Horng
authors.initials: CH
authors.lastname: Kuo
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Mei-Chin
authors.initials: MC
authors.lastname: Lai
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.|Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan.|Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Shih-Hsiung
authors.initials: SH
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000378:metabolism*; D001622:Betaine / Q000378:metabolism; D057232:Global Warming; D050938:Glycine N-Methyltransferase / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008697:Methane / Q000096:biosynthesis*; D017019:Methanosarcinaceae / Q000235:genetics / Q000502:physiology*; D008745:Methylation; D008958:Models, Molecular; D020125:Mutation, Missense; D064587:Osmoregulation / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D055049:Salt Tolerance / Q000235:genetics / Q000502:physiology; D053719:Tandem Mass Spectrometry
chemical_list: D019843:Archaeal Proteins; D010750:Phosphoproteins; D001622:Betaine; D050938:Glycine N-Methyltransferase; C456839:pyrrolysine; D008239:Lysine; D008697:Methane