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 constitute approximately 20% of integral plasma membrane protein. By using oligonucleotide primers based on partial amino acid sequences for polymerase chain reaction and screening of a spinach leaf cDNA library, we obtained 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 indicated that PM28A is the product of a single gene, and RNA gel blots showed that pm28a is ubiquitously expressed in the plant. In vivo phosphorylation of the 28-kD polypeptide(s), corresponding to PM28A and PM28B, was dependent on apoplastic water potential, suggesting a role in regulation of cell turgor for these putative aquaporins. In vitro, only one of the homologs, PM28A, was phosphorylated. Phosphorylation of PM28A occurred on Ser-274, seven amino acids from the C terminus of the protein, within a consensus phosphorylation site (Ser-X-Arg) for vertebrate 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+.
Johansson
I
I
Department of Plant Biochemistry, Lund University, Sweden.
Larsson
C
C
Ek
B
B
Kjellbom
P
P
D000595:Amino Acid Sequence; D020346:Aquaporins; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D015723:Gene Library; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D010766:Phosphorylation; D018515:Plant Leaves; D010940:Plant Proteins; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000502:physiology*; D014867:Water
D020346:Aquaporins; D017931:DNA Primers; D018076:DNA, Complementary; D008565:Membrane Proteins; C100932:PM28A protein, Spinacia oleracea; D010940:Plant Proteins; D011994:Recombinant Proteins; D014867:Water; D002118:Calcium