Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 293, Issue 44, Pages 17095-17106Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.003528
Keywords
H plus -ATPase; protein cross-linking; mass spectrometry (MS); protein-protein interaction; Arabidopsis thaliana
Categories
Funding
- National Science Foundation [MCB-1713899]
- Basic Energy Sciences Grant from the Department of Energy [DEFG02-88ER13938]
Ask authors/readers for more resources
In higher plants, a P-type proton-pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved, but direct chemical evidence for which ones are specifically interacting with the C terminus is lacking. In this study, we used in vivo cross-linking experiments with a photoreactive unnatural amino acid, p-benzoylphenylalanine, and tandem MS to obtain direct evidence that the C-terminal regulatory domain interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a head-to-tail organization of ATPase monomers in which the C-terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and it is consistent with prior studies suggesting that within the membrane the protein can be organized as homopolymers, including dimers, trimers, and hexamers.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available