DISTRIBUTION OF CHITIN SYNTHETASE AND VARIOUS MEMBRANE MARKER ENZYMESIN CHITOSOMES AND OTHER ORGANELLES OF THE SLIME MUTANT OF NEUROSPORA-CRASSA

The subcellular distribution of chitin synthetase was assessed in cell homogenates of the slime mutant of Neurospora crassa. The activities of several marker enzymes were monitored to establish the relationship between organelles containing chitin synthetase and other cytoplasmic organelles. Before cell breakage, the plasma membrane of the slime mu tant was radiolabeled with traces of [H-3]concanavalin A. Two major (I , II) and two minor (III, IV) bands of particles containing chitin syn thetase were detected after sedimentation of crude cell homogenates on a discontinuous sucrose gradient [1-4 h at 81,500g (R(av))]. The main population (I) consisted of microvesicles (chitosomes) which sediment ed more slowly than most other membranous organelles. The other major population (II) of chitin synthetase particles cosedimented with the p lasma membrane markers, [H-3]concanavalin A and a vanadate-sensitive A TPase. Despite differences in sedimentation velocity, the two major ch itin synthetase populations (I and II) eventually equilibrated at the same buoyant density (1.12 g/ml). One of the two minor bands (III) of chitin synthetase was associated with plasma membrane surface marker. Band IV, the smallest fraction with chitin synthetase activity was not characterized. A soluble, dialyzable, partially thermostable factor f ound in the cytosol of N. crassa enhanced the chitin synthetase activi ty of chitosomes (4.6 to 4.8-fold); to a lesser extent (about 2-fold) it also enhanced the activity of the other chitin synthetase populatio ns. This stimulatory factor found in the upper portion of the gradient s affected the levels of chitin synthetase activity detected, particul arly that of chitosomes in peak I. By a combination of centrifugation procedures, the subcellular populations of chitin synthetase were clea rly separated from other organelles with similar [endoplasmic reticulu m, d = 1.118, marker: phosphatidyl choline glyceride transferase] or d ifferent equilibrium densities [vacuole, d = 1.170 g/ml, marker: alpha -mannosidase; mitochondria, d = 1.175, marker: succinate dehydrogenase ]. The absence of markers for plasma membrane, mitochondria, and vacuo les from the chitosome population and the partial but unequivocal sepa ration of the endoplasmic reticulum marker from chitosomal chitin synt hetase provide additional verification that these microvesicles are di stinct subcellular structures and not fragments of other organelles. T he findings support the existence of a unique secretory pathway based on chitosome microvesicles as the main conveyors of chitin synthetase to the cell surface. (C) 1994 Academic Press, Inc.