The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an eleven gene family, constituting approximately two thirds of the total AHA mRNA in vegetative tissues. Collectively, AHA1 and AHA2 are essential for embryo development. In this report, we describe the translational fusion of a tandem affinity purification tag to the 5' end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryo lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions, and indicate a hexameric quatern... More
The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an eleven gene family, constituting approximately two thirds of the total AHA mRNA in vegetative tissues. Collectively, AHA1 and AHA2 are essential for embryo development. In this report, we describe the translational fusion of a tandem affinity purification tag to the 5' end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryo lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions, and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescued lines exhibit ca. 20% reduced root length, but no other visible phenotypic differences from wildtype are apparent. Quantitative analysis of plasma membrane and soluble proteomes identified a subtle proteomic phenotype in which several plasma membrane localized proteins show altered abundance in TAP-tagged AHA1 plants compared to wildtype. Using affinity purification coupled to mass spectrometry, we uniquely identified two AHA isoforms, AHA9 and AHA11, and additional candidate interacting proteins, which co-purified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 function and have shown that these plants can be used to purify AHA1 and identify in planta interacting proteins by mass spectrometry.
