A novel interaction partner for the C-terminus of Arabidopsis thaliana plasma membrane H+-ATPase (AHA1 isoform): site and mechanism of action on H+-ATPase activity differ from those of 14-3-3 proteins.
Using the two-hybrid technique we identified a novel protein whose N-terminal 88 amino acids (aa) interact with the C-terminal regulatory domain of the plasma membrane (PM) H+-ATPase from Arabidopsis thaliana (aa 847-949 of isoform AHA1). The corresponding gene has been named Ppi1 for Proton pump interactor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C-terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share significant similarity (50-70% at the aa level over stretches of 200-600 aa) to Ppi1. The PPI1 N-terminus, expressed in bacteria as a fusion protein with either GST or a His-tag, binds the PM H+-ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H+-ATPase activity. The effect of the His-tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C-terminal 10 kDa fragment. The His-tagged peptide binds also the trypsinised H+-ATPase. Altogether these results indicate that PPI1 N-terminus is able to modulate the PM H+-ATPase activity by binding to a site different from the 14-3-3 binding site and is located upstream of the trypsin cleavage site.
Dipartimento di Biologia L. Gorini, Sezione di Fisiologia e Biochimica delle Piante, Centro di Studio CNR-Biologia Cellulare e Molecolare delle Piante, c/o Dip. di Biologia, Via Celoria 26, 20133 Milan, Italy. piero.morandini@unimi.it
Piero
P
Morandini
Marco
M
Valera
Cristina
C
Albumi
Maria Cristina
MC
Bonza
Sonia
S
Giacometti
Giuseppe
G
Ravera
Irene
I
Murgia
Carlo
C
Soave
Maria Ida
MI
De Michelis
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D006027:Glycosides / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D006027:Glycosides; D007527:Isoenzymes; C469717:Ppi1 protein, Arabidopsis; D017494:Proton Pumps; D011993:Recombinant Fusion Proteins; C007808:fusicoccin; D014446:Tyrosine 3-Monooxygenase; D014357:Trypsin; D006180:Proton-Translocating ATPases