Isolation of prolegumin from developing pea seeds: Its binding to endomembranes and assembly into prolegumin hexamers in the protein storage vacuole

A fraction enriched in endoplasmic reticulum and Golgi membranes from developing cotyledons of Pisum sativum L. has proved to be a convenient source for the isolation of prolegumin, the precursor of the major 11S storage globulin of pea seeds. Two pro-proteins were isolated with molecular masses of 60 kDa and 75 kDa, respectively. A monoclonal antibody, designated 2B1, against prolegumin was raised using the in vitro immunization technique. This antibody recognizes the 60 kDa precursor polypeptide, but only the 20 kDa beta-subunit of mature legumin. Prolegumin, like the beta-subunit of the mature legumin, is a hydrophobic protein. After import into the protein storage vacuole, and after formation of the protein bodies trimeric 9S prolegumin assembles into 12S hexamers without prior processing of the precursor. Since prolegumin in vitro does not oligomerize into more than 9S trimers these results suggest that a protein-mediated assembly of 9S prolegumin trimers into 12S prolegumin hexamers probably occurs in the lumen of the protein storage vacuole. Prolegumin, but not mature legumin, binds very tightly to membranes. This property points to a possible way of identifying a putative prolegumin receptor.