Evidence That Glyoxysomal Malate Synthase Is Segregated by the Endoplasmic Reticulum

Abstract

At the onset of castor bean (Ricinus communis) germination, 76% of the cellular malate synthase activity of the endosperm tissue was located in the microsomal fraction, with the remainder in the glyoxysomal fraction. During later developmental stages, when rapid malate synthase synthesis was occurring, an increasing proportion of the enzyme was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into microsomal and glyoxysomal malate synthase in 2 day old endosperm tissue was followed by employing antiserum raised against glyoxysomal malate synthase to precipitate specifically the enzyme from KCl extracts of these organelle fractions. This experiment showed that microsomal malate synthase was labeled before the glyoxysomal enzyme. When such kinetic experiments were interrupted by the addition of an excess of unlabeled methionine, 35S-labeled malate synthase was rapidly lost from the microsomal fraction and was quantitatively recovered in the glyoxysomal fraction. Free cytoplasmic ribosomes were separated from bound ribosomes (rough microsomes) using endosperm tissue labeled with [35S]methionine or 14C-amino-acids. Nascent polypeptide chains were released from polysome fractions using a puromycin-high salt treatment, and radioactive malate synthase was shown to be exclusively associated with bound polysomes. Together these data establish that malate synthase is synthesized on bound ribosomes and vectorially discharged into the endoplasmic reticulum cisternae prior to its ultimate sequestration in glyoxysomes.