The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and a-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzymedigested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 'P-phospholipids, trichloroacetate-insoluble '4C, and trichloroacetate-soluble "4C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.In 1973, Weiss (35) and Subramanian et al. (28) reported the existence of an organelle in Neurospora crassa which sequestered basic amino acids. The organelle, called the "vesicle," was more dense than mitochondria (35) and invited comparison with the dense protease-containing particle, later called the vacuole (21), of Matile and co-workers (19). Because Weiss did not detect appreciable protease activity in his early work, and because the vacuole of N. crassa was characterized in certain conditions as a rather light organelle by other workers (17), the identity of the N. crassa vacuole remained open. In the meantime, Wiemken and his co-workers purified vacuoles from yeasts (11,38). Although yeast vacuoles are of low density and quite large, they contain most of the cellular basic amino acids, polyphosphates, proteases, a-mannosidase, and other hydrolytic enzymes (39). Recently, we showed (9) that the basic amino acids are located in the same dense organelle as the polyphosphates of N. crassa. We report here the purification of the N. crassa vacuole, defined by its amino acid and polyphosphate content, and its association with protease, a-mannosidase, and certain other enzyme activities. Purity was assessed by enzyme-marker studies and by a rationale of macromolecule labeling used in the purification of synaptic vesicles (6) of mammals. Osmotically induced density changes were shown to be the cause of the source of variation in density (1.06 to 1.35 g/cm3) reported by previous workers (16,17,19,21) for the N. crassa vacuole.(This paper is drawn from a dissertation by L.E.V. in partial fulfillment of the requirements for the Ph.D. in Biological Sciences, University of California, Irvine, 1980.)
MATERIALS AND METHODSStr...