Peripheral membrane proteins mediate binding of vacuolar storage proteins to membranes of the secretory pathway of developing pea cotyledons.

Abstract

In developing pea cotyledons, storage proteins are sorted via dense vesicles into the protein storage vacuole. Formation of these unique transport vesicles is characterized by aggregation of their cargo proteins. Protein sorting into dense vesicles is pH dependent. In order to gain insight into the molecular basis of storage protein sorting, a membrane binding assay was developed which allows for a detailed biochemical analysis of binding events. Employing this assay it was possible to show that storage proteins bind in a pH-dependent manner to the membranes of the secretory pathway with a pH optimum in the range of the lumenal pH of the Golgi cisternae. Through reconstitution experiments, it was possible to demonstrate further that this recruitment occurs via the interaction of peripheral rather than intrinsic membrane proteins. Results of co-immunoprecipitation experiments point to interactions between different storage proteins in the secretory system. These results are discussed in terms of the aggregation-mediated sorting of storage proteins into maturing dense vesicles.