Format

Send to

Choose Destination
Biochemistry. 2014 Jan 28;53(3):566-78. doi: 10.1021/bi401096m. Epub 2014 Jan 15.

Expression of a translationally fused TAP-tagged plasma membrane proton pump in Arabidopsis thaliana.

Author information

1
Department of Biochemistry, Biotechnology Center, University of Wisconsin , 425 Henry Mall, Madison, Wisconsin 53706, United States.

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.

PMID:
24397334
PMCID:
PMC3985734
DOI:
10.1021/bi401096m
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center