Ripening-associated proteolysis of a 27-kDa major intrinsic protein (MIP27) in tomato fruit

Major intrinsic proteins (MIPs) are believed to contribute towards the main tenance of structural integrity, osmoregulation, and responses to water and salt stresses in higher plants. In this work we identified a 27-kDa MTP (M IP27) in the microsomal membranes from tomato fruit using affinity purified antibodies to MIP27 from Beta vulgaris L. Sucrose gradient centrifugation analysis of microsomal membranes showed that MIP27 was associated with the plasma membrane and tonoplast fractions of tomato fruit. MIP27 aggregated t o a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis in the absen ce of dithiothreitol, a property characteristic of MIP proteins. MIP27 was degraded to a 25-kDa protein as tomato fruit progressed through the various stages of ripeness. The proteolytic degradation of MIP27 to the 25-kDa pro tein was also observed when microsomal membranes were treated with Pronase E. Treatment of microsomal membranes with thermolysin plus digitonin result ed in the complete degradation of MIP27. MIP27 was insensitive to treatment s with trypsin and carboxypeptidase Y. The proteolytic degradation of MIP27 may play a role in the structural integrity and textural properties of tom ato fruit during ripening.