Yeast mutants blocked in removing the methyl group of lanosterol at C-14. Separation of sterols by high-pressure liquid chromatography.

Sterols of a nystatin resistant mutant of the wild type parent of Saccharomyces cerevisiae were separated by a newly developed procedure involving high-pressure liquid chromatography and were identified. The mutant contained larger amounts of squalene and lanosterol (I) than the wild type, as well as 4,14-dimethylcholesta-8,24-dien-3beta-ol (II), 4,14-dimethylergosta-8,24(28)-dien-3beta-ol (III), and 14-methylergosta-8,24(28)-dien-3beta-ol (IV), which were not hitherto found in yeast. These results indicated a block in removal of the methyl group at C-14 of lanosterol. An ergosterol requiring derivative of the mutant which carried in addition a mutation in heme biosynthesis had the same sterols as the parent, but at one-third the concentration. The low level of sterols may be due to a requirement for a heme or cytochrome in oxygenation reactions between lanosterol and ergosterol.     

Azasterol inhibitors in yeast. Inhibition of the 24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferase of Saccharomyces cerevisiae by 23-azacholesterol.

The effects of 23-azacholesterol on sterol biosynthesis and growth of Saccharomyces cervisiae were examined. In the presence of 0.2, 0.5, and 1 micron 23-azacholesterol, aerobically-growing yeast produced a nearly constant amount of ergosta-5,7,22,24(28)-tetraenol (approx. 36% of total sterol) and slowly accumulated zymosterol with a concommitant decline in ergosterol synthesis. Growth and total sterol content of yeast cultures treated with 0.2-1 micron 23-azacholesterol were similar to that of the control culture. Yeast cultures treated with 5 and 10 micron 23-azacholesterol produced mostly zymosterol (58-61% of total sterol), while ergosta-5,7,22,24(28)-tetraenol production declined to less than 10% of total sterol. The observed changes in the distribution of sterols in treated cultures are consistent with inhibition of 24-methylene sterol 24(28)-sterol reductase (total inhibition at 1 micron 23-azacholesterol) and of 24-sterol methyltransferase (71% inhibition at 10 micron 23-azacholesterol). Yeast cultures treated with 10 micron 23-azacholesterol were found to contain 4,4-dimethylcholesta-8,14,24-trienol and 4alpha-methylcholesta-8,14,24-trienol, which were isolated and characterized for the first time.     

Isolation and characterization of yeast mutants blocked in mevalonic acid formation

Yeast mutants defective in beta-hydroxy-beta-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase have been isolated. Mutants impaired in acetoacetyl-CoA thiolase range into two linked complementation units, erg 10 A and erg 10 B. Mutants deficient in beta-hydroxy-beta-methylglutaryl-CoA synthase belong to two unlinked complementation groups, erg 11 and erg 13. In strictly anaerobic growth conditions, mutants impaired in beta-hydroxy-beta-methylglutaryl-CoA synthase require mevalonic acid in addition to sterol and oleic acid, pointing out the role of mevalonic acid in other physiological function than ergosterol precursor. Growth of mutants impaired in acetoacetyl-CoA thiolase cannot be recovered by mevalonic acid supplementation, suggesting a role of acetoacetyl-CoA or thiolase not linked to sterol pathway.     

Export of major cell surface proteins is blocked in yeast secretory mutants

The transport of newly synthesized proteins to the yeast cell surface has been analyzed by a modification of the technique developed by Kaplan et al. (Kaplan, G., C. Unkeless, and Z.A. Cohn, 1979, Proc. Natl. Acad. Sci. USA, 76:3824-3828). Cells metabolically labeled with (35)SO(4)(2-) are treated with trinitrobenzenesulfonic acid (TNBS) at 0 degrees C under conditions where cell-surface proteins are tagged with trinitrophenol (TNP) but cytoplasmic proteins are not. After fractionation of cells into cell wall, membrane and cytoplasmic samples, and solubilization with SDS, the tagged proteins are immunoprecipitated with anti-TNP antibody and fixed staphylococcus aureus cells. Analysis of the precipitates by SDS gel electrophoresis and fluorography reveals four major protein species in the cell wall (S(1)-S(4)), seven species in the membrane fraction (M(1)-M(7)), and no tagged proteins in the cytoplasmic fraction. Temperature-sensitive mutants defective in secretion of invertase and acid phosphatase (sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:204-215) are also defective in transport of the 11 major cell surface proteins at the nonpermissive temperature (37 degrees C). Export of accumulated proteins is restored in an energy- dependent fashion when secl cells are returned to a permissive temperature (24 degrees C). In wild-type cells the transit time for different surface proteins varies from less than 8 min to about 30 min. The asynchrony is developed at an early stage in the secretory pathway. All of the major cell wall proteins and many of the externally exposed plasma membrane proteins bind to concanavalin A. Inhibition of asparagine-linked glycosylation with tunicamycin does not prevent transport of several surface proteins.     

Inhibition of sterol synthesis by delta 5-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450

Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.     

A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway.

Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules. Using a sec6 mutant background to isolate vesicles, we have found that vacuolar protein sorting mutants that block an endosome-mediated route to the vacuole, including vps1, pep12, vps4, and a temperature-sensitive clathrin mutant, missort cargo normally transported by dense exocytic vesicles, such as invertase, into light exocytic vesicles, whereas transport of cargo specific to the light exocytic vesicles appears unaffected. Immunoisolation experiments confirm that missorting, rather than a changed property of the normally dense vesicles, is responsible for the altered density gradient fractionation profile. The vps41Delta and apl6Delta mutants, which block transport of only the subset of vacuolar proteins that bypasses endosomes, sort exocytic cargo normally. Furthermore, a vps10Delta sec6 mutant, which lacks the sorting receptor for carboxypeptidase Y (CPY), accumulates both invertase and CPY in dense vesicles. These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface. Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.     

Pseudomonas cepacia mutants blocked in the Entner-Doudoroff pathway.

Pseudomonas cepacia mutants deficient in either 6-phosphogluconate (6PGA) dehydratase (Edd-) or 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (Eda-) failed to utilize glucose or gluconate despite the prominence of of 6-phosphogluconate dehydrogenase (6PGAD) ii this bacterium and the potential for utilizing the pentose shunt suggested by its growth on ribitol and xylose. The Eda- strains grew normally on glucuronic acid, indicating that in P. cepacia its degradation does not depend upon KDPG aldolase as it does in Escherichia coli. Both 6PGA dehydratase and KDPG aldolase were inducible enzymes, with 6PGA rather than gluconate the apparent inducer. Edd- as well as Eda- strains were sensitive to growth inhibition by glucose, gluconate, fructose, and related carbohydrates when these substrates were present in combination with alternate carbon sources such as citrate or phthalate, presumably as a consequence of accumulation and toxicity of 6PGA, KDPG, or both. Edd- mutants were somewhat less sensitive to such inhibition than were Eda- strains. Certain derivatives of the Edd- strains we examined were able to utilize gluconate despite their deficiency of 6PGA dehydratase. Such mutants formed higher levels of 6PGAD than did the wild type. It is likely that the elevated levels of 6PGAD in these strains prevents accumulation of toxic levels of 6PGA that would otherwise result from a block in he Entner-Doudoroff pathway. The results suggest that P. cepacia can mutate to grow slowly on gluconate utilizing only the pentose shunt.     

Isolation of mutants blocked in calicheamicin biosynthesis.

Blocked mutants of Micromonospora echinospora defective in the production of the potent antitumor agent calicheamicin are described. Analysis of intermediates produced by blocked mutants indicates a branched biosynthetic pathway. Mutants that produced some, but not all, calicheamicin forms are also described.     

Structure-activity relationships of estrogens. Effects of 14-dehydrogenation and axial methyl groups at C-7, C-9 and C-11

Thirty compounds were evaluated in the rat for uterotropic effects, inhibition of gonadotropin release, and competitive displacement of (3H) estradiol-17 beta from uterine cytosolic preparations. 7 alpha-Methylestradiol-17 beta was 150% as active as estradiol-17 beta as an uterotropic agent. Estradiol-17 beta was the most active inhibitor of gonadotropin release. 11 beta-Methylestradiol-17 beta had 124% of the activity of estradiol-17 beta in displacing (3H) estradiol-17 beta from the "estrogen receptor." The 9 alpha-methyl group considerably decreased the potency of estrogens in any of the three assays. The 14-dehydro modification was advantageous only in the estradiol-17 beta 3-methyl ether series. Uterotropic activities and inhibition of gonadotropin release did not parallel. The best compound for inhibiting gonadotropin release, as compared to uterotropic activity, was estrone. The "estrogen receptor" assay data correlated fairly well with uterotropic assay data, but only for compounds having free 3-hydroxyl groups; even so, some exceptions were noted.     

Characteristics of Bacillus stearothermophilus mutants blocked in catabolic function.

With polyacrylamide disc gel electrophoresis and specific staining, it was demonstrated that one mutation involving the alcohol dehydrogenase of a double mutant of Bacillus stearothermophilus 1503 apparently prevented enzyme synthesis, and another lesion in the same organism resulted in synthesis of an inactive form of aconitase. Some properties of the double mutant and two fumarase mutants are discussed in relation to similar mutants derived from Bacillus subtilis.     

Methanol-dependent production of dihydroxyacetone and glycerol by mutants of the methylotrophic yeast Hansenula polymorpha blocked in dihydroxyacetone kinase and glycerol kinase

Summary Various factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250mM) and an additional substrate (0.5%, w/v) to replenish the xylulose-5-phosphate required for the assimilation reaction (DHA synthase) was essential for significant triose production by this double mutant. A number of sugars were tested as additional substrates and C₅ sugars gave the highest triose accumulation (ca. 20mM after 45h). Glucose was the poorest additional substrate and triose production only started after its exhaustion, which occurred in the first few hours. Other sugars were metabolized at a much lower rate and accumulation of trioses began right at the start of the experiments and gradually increased with time. The production rate of total trioses increased, and the relative amount of glycerol diminished with higher oxygen supply rates. The data suggest that conversion of DHA into glycerol, catalysed by reduced nicotine adenine dinucleotide (NADH)-dependent DHA reductase, is partly regulated via intracellular NADH levels. Further support for this hypothesis was obtained in experiments with antimycin A, an inhibitor of the electron transport chain. Addition of higher amounts of methanol and xylose, either by increasing the initial concentrations or by repeated addition of these substrates, resulted in considerably enhanced productivity and a switch towards glycerol formation. After reaching a level of approximately 25mM the DHA concentration remained constant while the glycerol level gradually increased with time. After an incubation period of 350 h, a total of 3.9 M methanol and 0.62 M xylose had been converted, which resulted in accumulation of 0.76 M trioses, mostly glycerol.Offprint requests to: L. Dijkhuizen     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

Induction of petite yeast mutants by membrane-active agents.

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.