Identification of an autoinhibitory domain in the C-terminal region of the plant plasma membrane H(+)-ATPase.
Proteolytic (trypsin) treatment removes a small terminal segment from the 100-kDa plant plasma membrane H(+)-ATPase. This results in activation of H+ pumping across the plasma membrane, suggesting that an inhibitory domain is located in one of the terminal regions of the enzyme (Palmgren, M.G., Larsson, C., and Sommarin, M. (1990) J. Biol. Chem. 265, 13423-13426). In order to identify the origin of the fragment released by trypsin, polyclonal antibodies were raised against the first 55 amino acids (N-terminal region), the last 99 amino acids (C-terminal region), and a portion of 150 amino acids in the central part of the enzyme as deduced from one of the H(+)-ATPase genes (PMA2) of Arabidopsis thaliana. The native, 100-kDa H(+)-ATPase was recognized by all three antisera in Western blots. By contrast, the approximately 90-kDa polypeptide appearing after trypsin treatment was only recognized by the antisera against the N-terminal and central region, but not by the antiserum against the C-terminal region, suggesting that the inhibitory domain is located in this part of the enzyme. To more closely determine the position of the inhibitory domain, three peptides representing conserved parts of the C-terminal region were synthesized (residues 861-888, 912-943, and 936-949 of the Arabidopsis (PMA2) sequence). Only one of the peptides (residues 861-888) affected H+ pumping by the trypsin-activated (approximately 90-kDa) enzyme. This peptide of 28 amino acids inhibited H+ pumping with an IC50 of about 15 microM, suggesting that the auto-inhibitory domain is located within the corresponding part of the C-terminal region.
Palmgren
M G
MG
Department of Plant Biochemistry, University of Lund, Sweden.
Sommarin
M
M
Serrano
R
R
Larsson
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin
D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases