Immunolocalization of a Cysteine Protease in Vacuoles, Vesicles, and Symbiosomes of Pea Nodule Cells

PsCYP15A is a cysteine protease from pea (Pisum sativum L.). It was first recognized as an up-regulated transcript in wilted shoots and subsequently in root nodules containing Rhizobium. Proteolytic activity of PsCYP15A in nodule extracts is now reported following immunopurification with polyclonal antiserum raised against recombinant antigen. Western-blot analysis indicated two forms of PsCYP15A, a pro-form (approximately 38 kD) and a mature form (approximately 30 kD). Both forms were present in most tissue samples, but only the mature form was isolated from cell-fractionated symbiosomes containing nitrogen-fixing bacteroids. Immunolabeling of nodule sections showed localization of PsCYP15A antigen in large vacuolar bodies, cytoplasmic vesicles, and the perisymbiont space. Immunolabeling of tissue sections from wilted shoots also indicated the presence of PsCYP15A in vacuoles and cytoplasmic vesicles. This protease may be involved in the adaptation to changes in cell turgor, both in wilted shoots and in nodule tissue. Additionally, the protease may be involved in protein turnover in the symbiosome compartment.