Identification and immunochemical characterization of a family of peroxisome membrane proteins (PMPs) in oilseed glyoxysomes

Abstract

Prior to this study the only antibodies available for characterizing peroxisome membrane proteins (PMPs) in plants were the antibodies raised against membranes isolated from castor bean endosperm glyoxysomes by Halpin et al. (Planta 179, 331-339 (1989)). We raised antibodies to four different nondenatured PMP complexes solubilized in 0.63 M aminocaproate/1% dodecylmaltoside from alkaline carbonate-washed, cucumber cotyledon glyoxysome membranes. The four complexes, approximately 290/270, 148, 128 and 67 kDa, were excised from 5 to 10% nondenaturing gradient gels, passively eluted from their homogenized gel slice, concentrated, then injected subcutaneously into rabbits. SDS-PAGE (10-15% gradient) of the total detergent-solubilized PMPs revealed six prominent membrane polypeptides: 73, 61, 52, 36, 30, and 22 kDa. The SDS-PMP composition of each nondenatured antigen was: PMP290/270-52, 30, 28 kDa; PMP148-30, 28, 26, 23, 22 kDa; PMP 128-73, 66, 36, 30, 23 kDa; PMP67-34, 30 kDa. These data indicated that several prominent as well as several minor polypeptides were common components of the PMP complexes. Three of the four antisera to the complexes were polyspecific, recognizing several of these common SDS polypeptides, whereas the fourth antiserum, anti-PMP67, was monospecific for PMP30. Cross-reactivities were evident with each antiserum to several of these SDS PMPs from castor bean, cotton and sunflower. Affinity-purified anti-PMP30 and anti-PMP73 antibodies specifically bound to the boundary membrane of cucumber glyoxysomes in cells examined by indirect, postembedment (LR White), immunocytochemistry. These, and the family of other antibodies produced in this study, provide specific molecular probes essential for elucidating biogenesis and discovering function(s) of the integral membrane proteins in oilseed glyoxysomes.