Biogenesis of Glyoxysomes

Abstract

Biosynthesis of isocitrate lyase, a tetrameric enzyme of the glyoxysomal matrix, was studied in Neurospora crassa, in which the formation of glyoxysomes was induced by a substitution of sucrose medium by acetate medium. 1 Translation of Neurospora mRNA in reticulocyte lysates yields a product which has the same apparent molecular weight as the subunit of the functional enzyme. Using N-formyl[³⁵S]methionyl tRNA^Met_f as a label, the translation product shows the same apparent size which indicates that the amino terminus has no additional ‘signal’-type sequence. 2 Read-out systems employing free and membrane-bound polysomes show that only free ribosomes are active in the synthesis of isocitrate lyase. 3 Isocitrate lyase synthesized in reticulocyte lysate is released into the supernatant and is soluble in a monomeric form. It interacts with Triton X-100 to form mixed micells in contrast to the functional tetrameric form. 4 Transfer of isocitrate lyase synthesized in vitro into isolated glyoxysomes is suggested by results of experiments in which supernatants from reticulocyte lysates are incubated with a particle fraction isolated from acetate-grown cells. No transfer occurs when particles from non-induced cells are employed. Resistance to added proteinase is used as a criterion for transmembrane transfer. The data support a post-translational transfer mechanism for isocitrate lyase. They suggest that isocitrate lyase passes through a cytosolic precurscr pool as a monomer and is transferred into glyoxysomes.