Isolation and characterization of the Neurospora crassa endoplasmic reticulum

Abstract

The endoplasmic reticulum from Neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. After differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane H+-ATPase. However, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. The lighter membrane fraction exhibited characteristics that have been associated with the endoplasmic reticulum in other organisms: (i) the inclusion of magnesium caused this light membrane fraction to shift to a higher density on the gradient; (ii) it was highly enriched in cytochrome c reductase, an endoplasmic reticulum marker in other systems; and (iii) the morphology of the light fraction with and without added magnesium was clearly distinguishable from that of the plasma membrane fraction by electron microscopy. A reinvestigation of the location of chitin synthetase confirmed its association with the plasma membrane fraction even after separation of the lighter fractions.