Distribution of Chitin Synthetase and Various Membrane Marker Enzymes in Chitosomes and Other Organelles of the Slime Mutant of Neurospora crassa

Abstract

Leal-Morales, C. A., Bracker, C. E., and Bartnicki-Garcia, S. 1994. Distribution of chitin synthetase and various membrane marker enzymes in chitosomes and other organelles of the slime mutant of Neurospora crassa. Experimental Mycology 18, 168-179. 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 mutant was radiolabeled with traces of [³H]concanavalin A. Two major (I, II) and two minor (III, IV) bands of particles containing chitin synthetase 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 sedimented more slowly than most other membranous organelles. The other major population (II) of chitin synthetase particles cosedimented with the plasma membrane markers, [³H]concanavalin A and a vanadate-sensitive ATPase. Despite differences in sedimentation velocity, the two major chitin 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 found in the cytosol of N. crassa enhanced the chitin synthetase activity 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 populations. This stimulatory factor found in the upper portion of the gradients affected the levels of chitin synthetase activity detected, particularly that of chirpspines in peak I. By a combination of centrifugation procedures, the subcellular populations of chitin synthetase were clearly separated from other organelles with similar [endoplasmic reticulum, d = 1.118, marker: phosphatidyl choline glyceride transferase] or different equilibrium densities [vacuole, d = 1.170 g/ml, marker: α-mannosidase; mitochondria, d = 1.175, marker: succinate dehydrogenase]. The absence of markers for plasma membrane, mitochondria, and vacuoles from the chitosome population and the partial but unequivocal separation of the endoplasmic reticulum marker from chitosomal chitin synthetase provide additional verification that these microvesicles are distinct subcellular structures and not fragments of other organelles. The findings support the existence of a unique secretory pathway based on chitosome microvesicles as the main conveyors of chitin synthetase to the cell surface.