title: Phosphoproteome exploration reveals a reformatting of cellular processes in response to low sterol biosynthetic capacity in Arabidopsis.
abstract: Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regulators of the biosynthetic flux from acyl-CoA toward phytosterols, we compared the membrane phosphoproteome of wild-type Arabidopsis thaliana and of a mutant being deficient in HMGR1. We performed a N-terminal labeling of microsomal peptides with a trimethoxyphenyl phosphonium (TMPP) derivative, followed by a quantitative assessment of phosphopeptides with a spectral counting method. TMPP derivatization of peptides resulted in an improved LC-MS/MS detection due to increased hydrophobicity in chromatography and ionization efficiency in electrospray. The phosphoproteome coverage was 40% higher with this methodology. We further found that 31 proteins were in a different phosphorylation state in the hmgr1-1 mutant as compared with the wild-type. One-third of these proteins were identified based on novel phosphopeptides. This approach revealed that phosphorylation changes in the Arabidopsis membrane proteome targets major cellular processes such as transports, calcium homeostasis, photomorphogenesis, and carbohydrate synthesis. A reformatting of these processes appears to be a response of a genetically reduced sterol biosynthesis.
authors.affiliation: Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique-Unité Propre de Recherche 2357, Université de Strasbourg , 28 rue Goethe, 67083 Strasbourg, France.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Heintz
authors.forename: Sebastien
authors.initials: S
authors.lastname: Gallien
authors.forename: Vincent
authors.initials: V
authors.lastname: Compagnon
authors.forename: Anne
authors.initials: A
authors.lastname: Berna
authors.forename: Masashi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yoshida
authors.forename: Toshiya
authors.initials: T
authors.lastname: Muranaka
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Christine
authors.initials: C
authors.lastname: Schaeffer
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Bach
authors.forename: Hubert
authors.initials: H
authors.lastname: Schaller
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000032:analysis / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010840:Phytosterols / Q000096:biosynthesis*; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D029681:Arabidopsis Proteins; D010750:Phosphoproteins; D010840:Phytosterols; D020543:Proteome; D006903:Hydroxymethylglutaryl CoA Reductases