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

Ole N. Jensen,Elena L. Rudashevskaya,Juanying Ye,Michael G. Palmgren,Anja T. Fuglsang

doi:10.1074/jbc.m111.307264

Ole N. Jensen, Elena L. Rudashevskaya + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m111.307264

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Abstract

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.

Highlights

Protein phosphorylation is an important posttranslational modification
To increase the number of phosphorylated sites detected, we treated the membranes with urea/thiourea and employed different phosphopeptide enrichment methods such as IMAC, TiO2, and CaPP followed by TiO2
New in Planta Phosphosites Identified in Arabidopsis Plasma Membrane Hϩ-ATPases—By employing complementary phosphopeptide enrichment methods, we have in this work identified 10 different in vivo phosphosites in different isoforms of the plasma membrane Hϩ-ATPase (Table 1), five of which have not been reported before, bringing the total number of phosphosites in this pump up to 11, which is substantially higher than reported for any other P-type ATPase

Summary

Introduction

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. 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

Results

Discussion

Conclusion