Phosphoproteome Exploration
Reveals a Reformatting
of Cellular Processes in Response to Low Sterol Biosynthetic Capacity
in <i>Arabidopsis</i>
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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 <i>Arabidopsis thaliana</i> 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 <i>hmgr1–1</i> 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 <i>Arabidopsis</i> 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