A GTP-binding protein regulates the activity of (1-->3)-beta-glucan synthase, an enzyme directly involved in yeast cell wall morphogenesis.

Synthesis of (1-->3)-beta-D-glucan, the major structural component of the yeast cell wall, is synchronized with the budding cycle. Membrane-bound, GTP-stimulated (1-->3)-beta-glucan synthase was dissociated by stepwise treatment with salt and detergents into two soluble fractions, A and B, both required for activity. Fraction A was purified about 800-fold by chromatography on Mono Q and Sephacryl S-300 columns. During purification, GTP binding to protein correlated with synthase complementing activity. A 20-kDa GTP-binding protein was identified by photolabeling in the purified preparation. This preparation no longer required GTP for activity, but incubation with another fraction from the Mono Q column (A1) led to hydrolysis of bound GTP to GDP with a concomitant return of the GTP requirement. Thus, fraction A1 appears to contain a GTPase-activating protein. These results show that the GTP-binding protein not only regulates glucan synthase activity but can be regulated in turn, constituting a potential link between cell cycle controls and wall morphogenesis.

Enzymatic hydrolysis and fermentation of corn for fuel alcohol.

The integration of enzyme saccharification with fermentation reduces the total time required to produce acceptable levels of ethanol. The use of a more concentrated mash (84.8 L total mash/bu corn) results in a 26.6% increase in ethanol productivity and a 21.4% increase in beer ethanol concentration compared to standard corn mash (96.6 L total mash/bu corn). Thus, the energy requirement and cost of distillation can be reduced. The addition of waste cola syrup at 30 g invert sugar/L total mash gave a 19% increase in ethanol concentration in the final beer and required only a small increase in the period of fermentation. Surplus laundry starch can replace 30-50% of the weight of corn normally used in fermentation without influencing ethanol production or the time required for fermentation. Both of these waste materials reduce the unit cost of ethanol and demonstrate the value of such substances in ethanol systems.

Hyphal tip growth in Achlya. I. Cytoplasmic organization.

Growing apices of Achlya ambisexualis Raper hyphae were examined by electron microscopy using cytochemical techniques. Apical vesicles can be grouped into two major classes based upon size and cytochemical reactions. Vesicles of the most prominent class are about 150 nm in diameter and possess contents which appear fibrous in thin section. This fibrous material reacts positively with the periodic acid-silver methenamine (PASM) cytochemical test for polysaccharides. Most of these same vesicles also display IDPase activity, and a smaller number display acid phosphatase activity. Vesicles of the second class are about 80 nm in diameter, and include coated vesicles and others which react positively for IDPase activity. They show a negative PASM reaction in contrast with the larger vesicles. Some of these smaller vesicles are stained by the phosphotungstic acid-chromic acid (PTA-CrO3) stain, whereas 150-nm vesicles are not. The source of at least some vesicles of both major classes appears to be the Golgi apparatus. It is proposed that the IDPase activity and carbohydrate content of the 150-nm cytoplasmic vesicles could serve as useful markers in their isolation.

Hyphal tip growth in Achlya. II. Subcellular localization of cellulase and associated enzymes.

The isolation and characterization of cellulase-containing membranes of Achlya ambisexualis Raper was attempted by differential and density gradient centrifugations. Maximum cellulase activity was found at an isopycnic density of 1.19 g/cm3, although some activity was found at other densities. A similar distribution of activity was shown by IDPase, ATPase, UDPG transferase, and by sedimentable carbohydrate. The coequilibration and steady enrichment of these activities during purification suggests their presence in a single type of subcellular particle. It was not possible to identify clearly the particle(s) in question from isolated fractions by electron microscopy, but when compared with the cytochemical localization of carbohydrate and IDPase in intact hypha, cytoplasmic vesicles with 150-mm diameters seem to be likely candidates.

A freeze-fracture study of hormone-induced branching in the fungus Achlya.

The induction of the male sexual organ primordia (antheridial hyphae) by the steriod hormone antheridiol in the water mold Achlya ambisexualis Raper requires the production and secretion of the enzyme cellulase. It is postulated that a localized secretion of cellulase produces a limited area of wall hydrolysis that is blown out into a lateral bleb by turgor pressure. Freeze-etch preparations show membrane profiles similar to those seen in other systems where exocytosis is occurring. Such a mechanism would provide the required localized secretion of cellulase. Water stress, imposed by polyethylene glycol, prevents the formation of antheridial hyphae, the secretion of cellulase and the expected membrane profiles. After a period of recovery from water stress antheridial hyphae are formed, cellulase secretion occurs and the expected membrane profiles are restored.

Preparation of coenocytes for freeze-etching.

Successful freeze-etching of a coenocyte has been accomplished with glutaraldehyde stabilization followed by infiltration with cryoprotectant. Hyphae of the coenocytic water mold Achlya were stabilized with 5% glutaraldehyde in phosphate buffer. Gradual infiltration by dropwise addition of the cryoprotectant (25% glycerol, 10% ethylene glycol, distilled water, v/v) is accomplished over a period of 8-10 hr on a shaker. Subsequent freeze-etching is carried out by standard procedures.

Sexual morphogenesis in Achlya: ultrastructural basis for the hormonal induction of antheridial hyphae.

Male strains of the water mold Achlya ambisexualis produce antheridial hyphae in response to the steroid hormone antheridiol. The antheridial hypha is postulated to be initiated through a localized wall softening with the enzyme cellulase. Freeze-etch studies of hormone-treated hyphae were conducted to determine if aggregates of vesicles are induced at the location of new antheridial hyphae. Localized aggregates of vesicles were found in conjunction with areas of wall thinning. These data suggest that the processes of vesiculation and secretion provide a mechanism for concentration of cellulase at the site of initiation of antheridial hyphae.

Hormonal control of sexual morphogenesis in achlya: dependence on protein and ribonucleic Acid syntheses.

The induction of the male sexual organ primordia (antheridial hyphae) by the steroid hormone antheridiol in the water mold Achlya ambisexualis requires both transcription and translation. Inhibition of either of these processes eliminates the expected increase in the production and release of the enzyme cellulase, which accompanies the formation of the antheridial hyphae.

Role of enzymatic wall-softening in plant morphogenesis: hormonal induction in Achlya.

Sexual hormone A, which induces antheridial branching in male strains of Achlya, also elicits a rise in cellulase. The peak of induced cellulase corresponds in time with the appearance of branches that are the male sexual organ primordia; only those strains that branch in response to the hormone show a concomitant rise in cellulase. The response to the hormone is inhibited by compounds that block protein synthesis, for example, p-fluorophenylalanine and puronmycin. Vegetative branching, induced by substrates such as casein hydrolysate, is also accompanied by a rise in cellulase.