Chitosomes from the wall-less "slime" mutant of Neurospora crassa.

Abstract

Cell-free extracts from the wall-less slime mutant of Neurospora crassa and the mycelium of wild type exhibit similar chitin synthetase properties in specific activity, zymogenicity and a preferential intracellular localization of chitosomes. The yield of chitosomal chitin synthetase from slime cells was essentially the same irrespective of cell breakage procedure (osmotic lysis or ballistic disruption)--an indication that chitosomes are not fragments of larger membranes produced by harsh (ballistic) disruption procedures. The plasma membrane fraction, isolated from slime cells treated with concanavalin A, contained only a minute portion of the total chitin synthetase of the fungus. Most of the activity was in the cytoplasmic fraction; isopycnic sedimentation of this fraction on a sucrose gradient yielded a sharp band of chitosomes with a buoyant density = 1.125 g/cm3. Approximately 76% of the total chitin synthetase activity of the slime mutant was recovered in the chitosome band. Because of their low density, chitosomes could be cleanly separated from the rest of the membranous organelles of the fungus. Apparently, the lack of a cell wall in the slime mutant is not due to the absence of either chitosomes or zymogenic chitin synthetase.