PT  - JOURNAL ARTICLE
AU  - Johnson, Kenneth D.
AU  - Chrispeels, Maarten J.
TI  - Tonoplast-Bound Protein Kinase Phosphorylates Tonoplast Intrinsic Protein
AID  - 10.1104/pp.100.4.1787
DP  - 1992 Dec 01
TA  - Plant Physiology
PG  - 1787--1795
VI  - 100
IP  - 4
4099  - http://www.plantphysiol.org/content/100/4/1787.short
4100  - http://www.plantphysiol.org/content/100/4/1787.full
SO  - Plant Physiol.1992 Dec 01; 100
AB  - Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases.