title: Expression of a translationally fused TAP-tagged plasma membrane proton pump in Arabidopsis thaliana.
abstract: The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report 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 embryonic 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 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified 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 functions and have shown that these plants can be used to purify AHA1 protein and to identify in planta interacting proteins by mass spectrometry.
authors.affiliation: Department of Biochemistry, Biotechnology Center, University of Wisconsin , 425 Henry Mall, Madison, Wisconsin 53706, United States.
authors.forename: Rachel B
authors.initials: RB
authors.lastname: Rodrigues
authors.forename: Gregorz
authors.initials: G
authors.lastname: Sabat
authors.forename: Benjamin B
authors.initials: BB
authors.lastname: Minkoff
authors.forename: Heather L
authors.initials: HL
authors.lastname: Burch
authors.forename: Thao T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sussman
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000096:biosynthesis*; D002462:Cell Membrane / Q000378:metabolism; D055785:Gene Knockdown Techniques; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification; D030821:Plants, Genetically Modified / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000096:biosynthesis*; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D029681:Arabidopsis Proteins; D007527:Isoenzymes; D006180:Proton-Translocating ATPases; C544444:AHA1 protein, Arabidopsis; C479903:AHA2 protein, Arabidopsis