Purification of vacuoles from Neurospora crassa.

Abstract

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...

Polyamine transport in Neurospora crassa

“…Cells were treated during growth with tracer levels of [3H]arginine to label the vacuolar pool of this amino acid (18), and, after transfer to Mops-glucose, they were loaded with 2 mM 14C-polyamine. After washes with 0.25 M NaCl in Mopsglucose, the cells were incubated for 5 min in Mops-glucose containing either 5 mM unlabeled polyamine, 7.5% n-butanol, or both (Table II).…”

An IP3-activated Ca2+ channel regulates fungal tip growth

“…The 80,000 g microsomal pellet was resuspended in 40-200 µl resuspension buffer (250 mM sucrose, 2.5 mM MES, 2.5 mM dithiotreitol adjusted to pH 7 with solid BTP) and layered onto a discontinuous three step sucrose gradient [5 ml of 16%, 29%, 39% (w/w) sucrose buffered to pH 7.2 with 5 mM MOPS/BTP] and centrifuged for 2 hours at 100,000 g at 4°C. This method was designed to partition the microsomal fraction into 'vacuolar' (16%) (Vaughn and Davis, 1981), ER (16/29%) (Borgeson and Bowman, 1983) and plasma membrane (29/39%) (Bowman et al, 1981) interfaces. The actual vacuolar membrane density has been reported to vary from 1.06 to 1.30 g cm -3 (Vaughn and Davis, 1981), depending on the details of homogenization and osmolarity.…”

“…This method was designed to partition the microsomal fraction into 'vacuolar' (16%) (Vaughn and Davis, 1981), ER (16/29%) (Borgeson and Bowman, 1983) and plasma membrane (29/39%) (Bowman et al, 1981) interfaces. The actual vacuolar membrane density has been reported to vary from 1.06 to 1.30 g cm -3 (Vaughn and Davis, 1981), depending on the details of homogenization and osmolarity. Because we used young germlings, which should not have had time to accumulate vacuolar polyphosphates and arginine, and homogenization at relatively low osmolarity (Vaughn and Davis, 1981), with EGTA present (Bowman and Bowman, 1982), we expect the vacuoles to have a low density, similar to plant vacuoles.…”

Phylogenetic considerations of neurosecretory granule contents: Role of nucleotides and basic hormone/transmitter packaging mechanisms.

“…Biochemical assays or ultrastructural hydrolytic digestion experiments will be necessary to positively identify the stained component as ATP. Polyphosphates have indeed been identified in yeast (OH-SUMI and ANRAKU, 1981;INDGE, 1968;URECH et al, 1978), algal (KUCHITSU et al, 1987) and mold (VAUGHN, 1981) vacuoles, where they serve to bind basic amino acids and act as a "cation trap" (DURR et al, 1979). Here, as in the mammalian chromaffin granules, polyphosphates, like ATP, can lower the osmotic pressure of yeast vacuoles that apparently store amino acids in the 1M range (DURR et al, 1979).…”

“…This property of amine sequestration may also have its origin in one-celled organisms, since yeasts (WIEMKEN and DURR, 1974;OHSUMI and ANRAKU, 1981;SATO et al, 1984a, b) and molds (VAUGHN and DAVIS, 1981) can effectively concentrate amino acids within cytoplasmic vacuoles and maintain a forty-fold gradient between the interior of the vacuole and the cytosol . The acid pH of the interior of the vacuole would protonate the basic amino acids and effectively "trap" the amino acids in an ionized state (DAVIS, 1958) within the membrane-bound compartment.…”