4 PUTATIVE, GLYOXYSOME MEMBRANE-PROTEINS ARE INSTEAD IMMUNOLOGICALLY-RELATED PROTEIN BODY MEMBRANE-PROTEINS

Previous research revealed that peroxisomes/glyoxysomes in plants, mammals, and yeasts possessed a prominent, integral peroxisome membrane protein (PMP) with a molecular mass in the range 21-26 kDa. We also found a major, low molecular mass polypeptide in the glyoxysome membrane fractions from four oilseed species: putative PMP26 in cotton (Gossypium hirsutum L.), PMP22 in cucumber (Cucumis sativus L.), PMP24.5 in sunflower (Helianthus annuus L.), and PMP24 in castor bean (Ricinus communis L.). Rabbit antiserum, produced against all the proteins which were solubilized from membranes recovered from isolated cotton glyoxysome fractions, recognized these four putative PMPs on Western blots as well as similar molecular mass polypeptides in mitochondria and protein bodies isolated from cotton and cucumber cotyledons. Postembedding, immunogold analyses of cotton and cucumber cotyledons, however, revealed that antibodies which were affinity-purified to the putative cotton glyoxysome 26-kDa polypeptide specifically bound to membranes of protein bodies, but not to membranes of glyoxysomes nor mitochondria. Antibodies to the bean tonoplast intrinsic protein (alpha-TIP), a known protein body membrane protein (PBMP), also recognized the four putative PMPs on Western blots and co-localized with anti-cotton PMP26 IgGs on thin sections to membranes of protein bodies. Intact protein bodies were not the source of the PBMPs in the oilseed organelle fractions; immunogold labeling revealed immunoreactive, small indiscrete vesicles interspersed among glyoxysomes and mitochondria in gradient fractions. Thus, putative prominent PMPs believed previously by us and others to be important in biogenesis and/or function of plant peroxisomes were discovered to be antigenically-related PBMPs of similar low molecular mass whose function in oilseeds has yet to be elucidated.