THE MAJOR INTEGRAL PROTEINS OF SPINACH LEAF PLASMA-MEMBRANES ARE PUTATIVE AQUAPORINS AND ARE PHOSPHORYLATED IN RESPONSE TO CA2+ AND APOPLASTIC WATER POTENTIAL

We show that homologs of the major intrinsic protein (MIP) family are major integral proteins of the spinach leaf plasma membrane and consti tute similar to 20% of integral plasma membrane protein, By using olig onucleotide primers based on partial amino acid sequences for polymera se chain reaction and screening of a spinach leaf cDNA library, we obt ained two full-length clones of MIP homologs (pm28a and pm28b). One of these clones, pm28a, was sequenced, and it encodes a protein (PM28A) of 281 amino acids with a molecular mass of 29.9 kD. DNA gel blots ind icated that PM28A is the product of a single gene, and RNA gel blots s howed that pm28a is ubiquitously expressed in the plant. In vivo phosp horylation of the 28-kD polypeptide(s), corresponding to PM28A and PM2 8B, was dependent on apoplastic water potential, suggesting a role in regulation of cell turgor for these putative aquaporins. In vitro, onl y one of the homologs, PM28A, was phosphorylated, Phosphorylation of P M28A occurred on Ser-274, seven amino acids from the C terminus of the protein, within a consensus phosphorylation site (Ser-X-Arg) for vert ebrate protein kinase C, In vitro phosphorylation of PM28A was due to a plasma membrane-associated protein kinase and was strictly dependent on submicromolar concentrations of Ca2+.