Phosphosite Mapping of P-type Plasma Membrane H+-ATPase in Homologous and Heterologous Environments

• Published in: The Journal of biological chemistry 2012-02, Vol.287 (7), p.4904-4913
• Main Authors: Rudashevskaya, Elena L., Ye, Juanying, Jensen, Ole N., Fuglsang, Anja T., Palmgren, Michael G.
• Format: Article
• Language: English
• Subjects: Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Cell Membrane - enzymology; Cell Membrane - genetics; Membrane Biology; Membrane Proteins; Phosphoproteomics; Phosphorylation; Phosphorylation - physiology; Plasma Membrane Proton Pumps; Protein Kinases; Protein Structure, Tertiary; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - genetics; Proton-Translocating ATPases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - genetics; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.307264

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H+-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H+-ATPase to protein kinase action. Background: Protein phosphorylation is an important posttranslational modification. Results: Both in planta and when expressed in yeast, the P-type proton pump is phosphorylated at multiple new positions at its terminal regulatory domains. Conclusion: Multiple methods for phosphopeptide enrichment are required for complete phosphosite mapping. Significance: This work provides a surprising example of functional conservation of protein kinase action between plants and yeast.