Synthesis and posttranslational segregation of glyoxysomal isocitrate lyase from castor bean endosperm.
Total polyadenylated RNA isolated from 3-day-old germinating castor bean endosperm was translated in the rabbit reticulocyte lyase cell-free protein-synthesizing system in either the presence or absence of canine pancreatic microsomal vesicles. Isocitrate lyase was immunoprecipitated from the translational products using rabbit antibodies raised against the purified glyoxysomal enzyme. Isocitrate lyase synthesized in vitro had the same apparent molecular weight as the authentic glyoxysomal enzyme irrespective of whether synthesis occurred in the presence or absence of microsomes. Furthermore, in contrast to several proteins encoded by mRNA isolated from maturing seed endosperm tissue, isocitrate lyase was not cotranslationally segregated into the lumen of the microsomal vesicles. Intact 3-day-old endosperm tissue was labelled in vivo by adding [35S]methionine and at various times the tissue was homogenized and the cellular organelles fractionated. Separated fractions were assayed for newly synthesized isocitrate lyase. Immunoreactive product was initially detected in the cytosolic fraction and subsequently began to accumulate in glyoxysomes. The results indicate that glyoxysomal isocitrate lyase is synthesized on free polysomes and released into the cytosol. Transfer to the matrix of the glyoxysome involves a posttranslational membrane translocation step which is not accompanied by proteolytic cleavage of the polypeptide chain.
D000818:Animals; D000906:Antibodies / Q000032:analysis; D002367:Ricinus communis / Q000201:enzymology*; D004285:Dogs; D066298:In Vitro Techniques; D007522:Isocitrate Lyase / Q000302:isolation & purification / Q000378:metabolism*; D008830:Microbodies / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D009940:Organoids / Q000201:enzymology*; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D010947:Plants, Toxic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012278:Ricinus / Q000201:enzymology*
D000906:Antibodies; D012333:RNA, Messenger; D007652:Oxo-Acid-Lyases; D007522:Isocitrate Lyase