Biogenesis of glyoxysomes. Synthesis and intracellular transfer of isocitrate lyase.

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-tRNAfMet 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 precursor pool as a monomer and is transferred into glyoxysomes.