Phosphosite mapping of P-type plasma membrane H⁺-ATPase in homologous and heterologous environments

TY - JOUR

T1 - Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments

AU - Rudashevskaya, Elena

AU - Ye, Juanying

AU - Jensen, Ole Nørregaard

AU - Fuglsang, Anja Thoe

AU - Palmgren, Michael Broberg

PY - 2012/2/10

Y1 - 2012/2/10

N2 - 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.

AB - 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.

U2 - 10.1074/jbc.M111.307264

DO - 10.1074/jbc.M111.307264

M3 - Journal article

C2 - 22174420

SN - 0021-9258

VL - 287

SP - 4904

EP - 4913

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 7

ER -