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Nitrogen-responsive control of Gln3 localization is implemented through TorC1-dependent (rapamycin-responsive) and TorC1-independent (nitrogen catabolite repression-sensitive and methionine sulfoximine (Msx)-responsive) regulatory pathways. We previously demonstrated amino acid substitutions in a putative Gln3 α-helix(656-666), which are required for a two-hybrid Gln3-Tor1 interaction, also abolished rapamycin responsiveness of Gln3 localization and partially abrogated cytoplasmic Gln3 sequestration in cells cultured under nitrogen-repressive conditions. Here, we demonstrate these three characteristics are not inextricably linked together. A second distinct Gln3 region (Gln3(510-589)) is specifically required for rapamycin responsiveness of Gln3 localization, but not for cytoplasmic Gln3 sequestration under repressive growth conditions or relocation to the nucleus following Msx addition. Aspartate or alanine substitution mutations throughout this region uniformly abolish rapamycin responsiveness. Contained within this region is a sequence with a predicted propensity to form an α-helix(583-591), one side of which consists of three hydrophobic amino acids flanked by serine residues. Substitution of aspartate for even one of these serines abolishes rapamycin responsiveness and increases rapamycin resistance without affecting either of the other two Gln3 localization responses. In contrast, alanine substitutions decrease rapamycin resistance. Together, these data suggest that targets in the C-terminal portion of Gln3 required for the Gln3-Tor1 interaction, cytoplasmic Gln3 sequestration, and Gln3 responsiveness to Msx addition and growth in poor nitrogen sources are distinct from those needed for rapamycin responsiveness.

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. Background: Five different conditions have been employed interchangeably to down-regulate TorC1. Results: Four of the five conditions exhibit different, hierarchially related phosphatase requirements. Gln3 responses to long-term nitrogen starvation and Msx treatment exhibit the same phosphatase requirements. Conclusion: Previous conditions for down-regulating TorC1 are not physiologically equivalent. Significance: The cellular responses to varying nitrogen supplies occur via multiple, distinguishable regulatory pathways.

The GATA family transcription activator, Gln3 responds to the nitrogen requirements and environmental resources of the cell. When rapidly utilized, "good" nitrogen sources, e.g., glutamine, are plentiful, Gln3 is completely sequestered in the cytoplasm, and the transcription it mediates is minimal. In contrast, during nitrogen-limiting conditions, Gln3 quickly relocates to the nucleus and activates transcription of genes required to scavenge alternative, "poor" nitrogen sources, e.g., proline. This physiological response has been designated nitrogen catabolite repression (NCR). Because rapamycin treatment also elicits nuclear Gln3 localization, TorC1 has been thought to be responsible for NCR-sensitive Gln3 regulation. However, accumulating evidence now suggests that GATA factor regulation may occur by two separate pathways, one TorC1-dependent and the other NCR-sensitive. Therefore, the present experiments were initiated to identify Gln3 amino acid substitutions capable of dissecting the individual contributions of these pathways to overall Gln3 regulation. The rationale was that different regulatory pathways might be expected to operate through distinct Gln3 sensor residues. We found that C-terminal truncations or amino acid substitutions in a 17-amino acid Gln3 peptide with a predicted propensity to fold into an α-helix partially abolished the ability of the cell to sequester Gln3 in the cytoplasm of glutamine-grown cells and eliminated the rapamycin response of Gln3 localization, but did not adversely affect its response to limiting nitrogen. However, overall wild type control of intracellular Gln3 localization requires the contributions of both individual regulatory systems. We also found that Gln3 possesses at least one Tor1-interacting site in addition to the one previously reported.

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Δ22Δ, tpd3Δ, or cdc55Δ cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.

The Tor1,2 protein kinases globally influence many cellular processes including nitrogen-responsive gene expression that correlates with intracellular localization of GATA transcription activators Gln3 and Gat1/Nil1. Gln3-Myc13 and Gat1-Myc13 are restricted to the cytoplasm of cells provided with good nitrogen sources, e.g. glutamine. Following the addition of the Tor1,2 inhibitor, rapamycin, both transcription factors relocate to the nucleus. Gln3-Myc13 localization is highly dependent upon Ure2 and type 2A-related phosphatase, Sit4. Ure2 is required for Gln3 to be restricted to the cytoplasm of cells provided with good nitrogen sources, and Sit4 is required for its location to the nucleus following rapamycin treatment. The paucity of analogous information concerning Gat1 regulation prompted us to investigate the effects of deleting SIT4 and URE2 on Gat1-Myc13 localization, DNA binding, and NCR-sensitive transcription. Our data demonstrate that Tor pathway control of NCR-responsive transcription bifurcates at the regulation of Gln3 and Gat1. Gat1-Myc13 localization is not strongly influenced by deleting URE2, nor is its nuclear targeting following rapamycin treatment strongly dependent on Sit4. ChIP experiments demonstrated that Gat1-Myc13 can bind to the DAL5 promoter in the absence of Gln3. Gln3-Myc13, on the other hand, cannot bind to DAL5 in the absence of Gat1. We conclude that: (i) Tor pathway regulation of Gat1 differs markedly from that of Gln3, (ii) nuclear targeting of Gln3-Myc13 is alone insufficient for its recruitment to the DAL5 promoter, and (iii) the Tor pathway continues to play an important regulatory role in NCR-sensitive transcription even after Gln3-Myc13 is localized to the nucleus.

Saccharomyces cerevisiae selectively utilizes good nitrogen sources in preference to poor ones by down-regulating transcription of genes encoding proteins that transport and degrade poor nitrogen sources when excess nitrogen is available. This regulation is designated nitrogen catabolite repression (NCR). When cells are transferred from a good to a poor nitrogen source (glutamine to proline) or treated with rapamycin, an inhibitor of the protein kinases Tor1/2, Gln3 (NCR-sensitive transcription activator) moves from the cytoplasm into the nucleus. Gln3 re-accumulates in the cytoplasm when cells are returned to a good nitrogen source. However, Gln3 is not uniformly distributed in the cytoplasm. Such non-uniform distribution could result from a variety of interactions including association with a cytoplasmic vesicular system or components of the cytoskeleton. We used latrunculin, a drug that disrupts the actin cytoskeleton by inhibiting actin polymerization, to determine whether the actin cytoskeleton participates in intracellular Gln3 movement. Latrunculin-treatment prevents nuclear accumulation of Gln3 and NCR-sensitive transcription in cells transferred from ammonia to proline medium but does not prevent its accumulation in the cytoplasm of cells transferred from proline to glutamine medium. In contrast, rapamycin-induced nuclear accumulation of Gln3 is not demonstrably affected by latrunculin treatment. These data indicate the actin cytoskeleton is required for nuclear localization of Gln3 in response to limiting nitrogen but not rapamycin-treatment. Therefore, the actin cytoskeleton either participates in the response of Gln3 intracellular localization to nitrogen limitation before Tor1/2, or Tor1/2 inhibition only mimics the outcome of nitrogen limitation rather than directly regulating it.

Retrograde genes of Saccharomyces cerevisiae encode the enzymes needed to synthesize alpha-ketoglutarate, required for ammonia assimilation, when mitochondria are damaged or non-functional because of glucose fermentation. Therefore, it is not surprising that a close association exists between control of the retrograde regulon and expression of nitrogen catabolic genes. Expression of these latter genes is nitrogen catabolite repression (NCR)-sensitive, i.e. expression is low with good nitrogen sources (e.g. glutamine) and high when only poor (e.g. proline) or limiting nitrogen sources are available. It has been reported recently that both NCR-sensitive and retrograde gene expression is negatively regulated by glutamine and induced by treating cells with the Tor1/2 inhibitor, rapamycin. These conclusions predict that NCR-sensitive and retrograde gene expression should respond in parallel to nitrogen sources, ranging from those that highly repress NCR-sensitive transcription to those that elicit minimal NCR. Because this prediction did not accommodate earlier observations that CIT2 (a retrograde gene) expression is higher in glutamine than proline containing medium, we investigated retrograde regulation further. We show that (i) retrograde gene expression correlates with intracellular ammonia and alpha-ketoglutarate generated by a nitrogen source rather than the severity of NCR it elicits, and (ii) in addition to its known regulation by NCR, NAD-glutamate dehydrogenase (GDH2) gene expression is down-regulated by ammonia under conditions where NCR is minimal. Therefore, intracellular ammonia plays a pivotal dual role, regulating the interface of nitrogen and carbon metabolism at the level of ammonia assimilation and production. Our results also indicate the effects of rapamycin treatment on CIT2 transcription, and hence Tor1/2 regulation of retrograde gene expression occur indirectly as a consequence of alterations in ammonia and glutamate metabolism.

The apolipoprotein (apo) E receptor-2 (apoER2) is a member of the low density lipoprotein receptor gene family and an important regulator of neuronal migration. It acts as a receptor for the signaling factor Reelin and provides positional cues to neurons that migrate to their proper position in the developing brain. Besides brain formation defects, apoER2-deficient mice also exhibit male infertility. The role of the receptor in male reproduction, however, remained unclear. Here we demonstrate that apoER2 is highly expressed in the initial segment of the epididymis, where it affects the functional expression of clusterin and phospholipid hydroperoxide glutathione peroxidase (PHGPx), two proteins required for sperm maturation. Reduced PHGPx expression in apoER2 knockout mice results in the inability of the sperm to regulate the cell volume and in abnormal sperm morphology and immotility. Because insufficient expression of PHGPx is a major cause of infertility in men, these findings not only highlight an important new function for apoER2 that is unrelated to neuronal migration, but they also suggest a possible role for apoER2 in human infertility.

Ure2, the protein that negatively regulates GATA factor (Gln3, Gat1)-mediated transcription in Saccharomyces cerevisiae, possesses prion-like characteristics. Identification of metabolic and environmental factors that influence prion formation as well as any activities that prions or prion precursors may possess are important to understanding them and developing treatment strategies for the diseases in which they participate. Ure2 exhibits primary sequence and three-dimensional homologies to known glutathione S-transferases. However, multiple attempts over nearly 2 decades to demonstrate Ure2-mediated S-transferase activity have been unsuccessful, leading to the possibility that Ure2 may well not participate in glutathionation reactions. Here we show that Ure2 is required for detoxification of glutathione S-transferase substrates and cellular oxidants. ure2 Delta mutants are hypersensitive to cadmium and nickel ions and hydrogen peroxide. They are only slightly hypersensitive to diamide, which is nitrogen source-dependent, and minimally if at all hypersensitive to 1-chloro-2,4-dinitrobenzene, the most commonly used substrate for glutathione S-transferase enzyme assays. Therefore, Ure2 shares not only structural homology with various glutathione S-transferases, but ure2 mutations possess the same phenotypes as mutations in known S. cerevisiae and Schizosaccharomyces pombe glutathione S-transferase genes. These findings are consistent with Ure2 serving as a glutathione S-transferase in S. cerevisiae.

Regulated intracellular localization of Gln3, the transcriptional activator responsible for nitrogen catabolite repression (NCR)-sensitive transcription, permits Saccharomyces cerevisiae to utilize good nitrogen sources (e.g. glutamine and ammonia) in preference to poor ones (e.g. proline). During nitrogen starvation or growth in medium containing a poor nitrogen source, Gln3 is nuclear and NCR-sensitive transcription is high. However, when cells are grown in excess nitrogen, Gln3 is localized to the cytoplasm with a concomitant decrease in gene expression. Treating cells with the Tor protein inhibitor, rapamycin, mimics nitrogen starvation. Recently, carbon starvation has been reported to cause nuclear localization of Gln3 and increased NCR-sensitive transcription. Here we show that nuclear localization of Gln3 during carbon starvation derives from its indirect effects on nitrogen metabolism, i.e. Gln3 does not move into the nucleus of carbon-starved cells if glutamine rather than ammonia is provided as the nitrogen source. In addition, these studies have clearly shown Gln3 is not uniformly distributed in the cytoplasm, but rather localizes to punctate or tubular structures. Analysis of these images by deconvolution microscopy suggests that Gln3 is concentrated in or associated with a highly structured system in the cytosol, one that is possibly vesicular in nature. This finding may impact significantly on how we view (i) the mechanism by which Tor regulates the intracellular localization of Gln3 and (ii) how proteins move into and out of the nucleus.

Previous studies have shown that (i) Dal81p and Dal82p are required for allophanate-induced gene expression inSaccharomyces cerevisiae; (ii) the cis-acting element mediating the induced transcriptional response to allophanate is a dodecanucleotide, UIS ALL; and (iii) Dal82p binds specifically to UIS ALL. Here we show that Dal82p is localized to the nucleus and parallels movement of the DNA through the cell cycle. Deletion analysis of DAL82identified and localized three functional domains. Electrophoretic mobility shift assays identified a peptide (consisting of Dal82p amino acids 1–85) that is sufficient to bind a DNA fragment containingUIS ALL. LexA-tethering experiments demonstrated that Dal82p is capable of mediating transcriptional activation. The activation domain consists of two parts: (i) an absolutely required core region (amino acids 66–99) and (ii) less well defined regions flanking residues 66–99 that are required for full wild-type levels of activation. The Dal82p C terminus contains a predicted coiled-coil motif that down-regulates Dal82p-mediated transcriptional activation.

Saccharomyces cerevisiae can use urea as sole nitrogen source by degrading it in two steps (urea carboxylase and allophanate hydrolase) to ammonia and carbon dioxide. We previously demonstrated that: 1) the enzymatic functions required for degradation are encoded in two tightly linked genetic loci and 2) pleiotropic mutations each resulting in the loss of both activities are found in both loci. These and other observations led to the hypothesis that urea degradation might be catalyzed by a multifunctional polypeptide. Waheed and Castric (1977) J. Biol. Chem. 252, 1628-1632), on the other hand, purified urea amidolyase from Candida utilis and reported it to be a tetramer composed of nonidentical 70- and 170-kilodalton subunits. To resolve the differing views of urea amidolyase structure, we purified the protein using rapid methods designed to avoid proteolytic cleavage. Application of these methods resulted in the isolation of a single, inducible and repressible, 204-kilodalton species. We observed no evidence for the existence of nonidentical subunits. A similar inducible, high molecular weight species was also detected in C. utilis. These biochemical results support our earlier hypothesis that urea degradation is carried out in yeast by an inducible and repressible protein composed of identical, multifunctional subunits.

We report here the isolation and structure of a new repeated DNA element, tau. This element, from Saccharomyces cerevisiae, is 371 base pairs long and is flanked on either end by the same invertedly repeated sequence found at the ends of some Ty and sigma elements in yeast, copia elements in Drosophila and spleen necrosis virus. The tau inverted repeats are themselves flanked by a 5-base pair directly repeated genomic sequence that is present only once in a cognate tau-allele. These structural characteristics, the presence of multiple copies of tau in the genome, and the isolation of tau+ and tau- allelic pairs suggest that tau may be capable of transposition either alone or in association with some larger element. Detailed sequence analysis of the tau, sigma, and delta elements revealed that all three contain significant regions of homology, suggesting that they are probably members of a single family derived from a common progenitor.

PUT cells were selected from the XD line of cultured tobacco cells (Nicotiana tabacum L. cv. Xanthi-nc) for the ability to utilize putrescine as sole nitrogen source. Previous work had indicated that hydroxycinnamoylputrescines (principally caffeoylputrescine) and 4-amino-n-butyric acid (GABA) are obligatory intermediates in the assimilation of putrescine by PUT cells. The apparent absence in these cells of diamine or polyamine oxidase and pyrroline dehydrogenase, enzymes which catalyze putrescine oxidation in some plant species, led us to propose the following pathway for putrescine oxidation in PUT cells: putrescine----hydroxycinnamoylputrescine----hydroxycinnamoyl - 4-aminobutyraldehyde----hydroxycinnamoyl-GABA----GABA. We tested the hypothesis by looking for the predicted compound, caffeoyl-GABA. A chemical synthesis was developed, and chromatographic and mass spectroscopic procedures were devised for identifying the compound in extracts of cells and plant tissues. Caffeoyl-GABA was found in extracts of PUT cells in micromolar concentrations but was not present in XD cells. Thus, its occurrence in PUT cells appears to be a direct result of selection for the ability to catabolize putrescine. Caffeoyl-GABA has the same distribution in tobacco plants as caffeoylputrescine, i.e. flower buds greater than open flowers greater than floral leaves, green fruit; absent in vegetative tissues.

Vitamin K participates in the post-translational carboxylation of peptide-bound glutamate to form the gamma-carboxy-glutamate residues of prothrombin. The reaction requires reduced vitamin K, bicarbonate, oxygen, and a membrane-bound carboxylase. The active species of "CO2," i.e. CO2 or HCO3-, utilized in this carboxylation was determined by the low temperature method of Filmer and Cooper ((1970) J. Theor. Biol. 29, 131-145), taking advantage of the fact that menaquinone-2, in contrast to phylloquinone, is very active at 10 degrees. Microsomes from livers of vitamin K-deficient rats, were incubated in the presence of cycloheximide, avidin, NADH, menaquinone-2, 1 mM acetazolamide (to inhibit carbonic anhydrase), and either 14CO2 or H14CO3-. At 1-min intervals aliquots were removed from the reaction mixture. gamma-Carboxyglutamate was isolated from these samples by ion exchange chromatography after alkaline hydrolysis. After 1 min the incorporation of 14CO2 into gamma-carboxyglutamate was 8 to 10 times as great as that found with H14CO3-. When the carbonic anhydrase inhibitor was omited (with or without addition of exogenous carbonic anhydrase) the two incorporation curves approximated each other at a rate near that exhibited by bicarbonate alone. Similar results were obtained in a microsomal carboxylase system solubilized with Triton X-100. It is concluded that CO2 is the active species of "CO2" initially participating in the vitamin K-dependent carboxylation of preprothrombin and that neither ATP nor biotin is required for the reaction.

Using a well defined system (lac operon) composed of single species of messenger RNA and DNA, the chemistry of the hybridization reaction has been studied. The reaction appears to be "kinetically irreversible." Hybridization techniques predicated upon this irreversibility permit the recovery of the transcription products of either the proximal (Z) or distal (Y-A) cistrons of the lacoperon. In addition a 500- fold increase in the level of the lac-specific RNA was observed upon induction of the operon. This compares favorably with the increase in enzyme concentration.

The effects of induction and of transient and catabolite repression on lac-specific RNA in cells of Escherichia coli were investigated. The rate of lac-specific RNA synthesis increases linearly for 3 min after induction and then becomes constant. The accumulation of RNA complementary to the promoter proximal lac Z gene begins before accumulation of RNA complementary to the promoter distal lac Y-A genes. When transient repression is imposed upon the cells by the addition of glucose, the lac Z-specific RNA begins to decay before the lac Y-A-specific RNA. Catabolite repression reduces the rate of formation of lac-specific RNA relative to the rate of formation of bulk RNA. Both lac Z-specific RNA and lac Y-A-specific RNA are equally affected. These results confirm the concept that the sole effect of transient repression and of catabolite repression is the inhibition of the initiation of RNA synthesis at the lac promoter site.

The demonstration that the reactions of β oxidation and the glyoxylate cycle occur in the glyoxysome, a cellular organelle isolated from castor bean endosperm, has raised the question of the intracellular location of the enzymatic activation of free fatty acids in this tissue. Glyoxysomes, isolated on sucrose gradients, have been shown to contain a thiokinase which, in the presence of CoA, MgCl2, and ATP (GTP is ineffective), activates free fatty acids to their cognate fatty acyl-CoA derivatives. The thiokinase is specific for fatty acids of chain length greater than C-10. An acetate thiokinase was also found in glyoxysomes. It is specific for ATP and is more tightly bound to the organelles than the long chain thiokinase. Although the long chain thiokinase is easily solubilized, the data show that the intact glyoxysomes can convert free fatty acids to their CoA derivatives and oxidize them to acetyl-CoA.

Previous studies with propionyl coenzyme A carboxylase and with phosphoenolpyruvate carboxylase using 18O-labeled bicarbonate have indicated that bicarbonate is the reactive species in these fixations of "CO2." We have investigated the species of CO2 in reactions catalyzed by pyruvate carboxylase, P-enolpyruvate carboxykinase, and P-enolpyruvate carboxytransphosphorylase. Since propionyl-CoA carboxylase and pyruvate carboxylase are biotin enzymes, they would be expected to have similar mechanisms. Likewise, the reactions catalyzed by P-enolpyruvate carboxykinase and carboxy transphosphorylase are, in some respects, similar to that of P-enolpyruvate carboxylase, and it has been suggested that bicarbonate might be the reactant in each case. By means of radiochemical and spectrophotometric techniques, we have obtained evidence that the active species in the carboxykinase and carboxytransphosphorylase reactions is CO2 and not bicarbonate. Bicarbonate appears to be the active species in the pyruvate carboxylase reaction, in conformity with the results obtained with propionyl-CoA carboxylase.

Saccharomyces cerevisiae can utilize urea as a sole nitrogen source; this compound is degraded to CO2 and NH3 by a multienzyme complex (Whitney, P. A., and Cooper, T. G. (1972) Biochem. Biophys. Res. Commun. 49, 45) composed of urea carboxylase and allophanate hydrolase. Since these activities are present in cultures grown in media containing urea, arginine, or a purine base, the nature of the inducer was investigated. The data presented here indicate that it is the intermediate compound, allophanate, which is the inducer of both urea carboxylase and allophanate hydrolase. This conclusion is based on the observation that: (a) urea induces both activities in a wild type strain, (b) urea has no effect on the basal levels of allophanate hydrolase produced by mutants lacking urea carboxylase, and (c) urea has no effect on the high constitutive level of urea carboxylase produced by a mutant lacking allophanate hydrolase. Consistent with this view is the observation that a urea analogue, formamide, cannot induce allophanate hydrolase in a strain lacking urea carboxylase activity, whereas an allophanate analogue, hydantoic acid, is able to serve as an inducer in such a strain. Both of these compounds are able to effect the induction of both activities in a wild type strain. Mutants blocked in either arginase or urea carboxylase were also used to show that the derepression of allophanate hydrolase following the onset of nitrogen starvation is, in fact, internal induction, and is contingent upon the ability of the cells to produce the inducer, allophanate, from internal arginine and urea pools.

Crude particulate preparations from castor bean endosperm contain most of the β oxidation activity present in initial extracts. Measurements on organelles separated by sucrose density centrifugation of the crude particles show that glyoxysomes (density 1.25) contain more than 80% of the particulate β oxidation activity, with virtually none in the mitochondria (density 1.19). Thiolase shows a similar distribution. Glyoxysomes do not oxidize NADH, but O2 is required for β oxidation. Addition of palmitoyl coenzyme A to glyoxysomes results in O2 uptake, NADH accumulation, and acetyl-CoA production, in a 0.5:1:1 stoichiometry. Cyanide has no effect on NADH accumulation, but doubles the rate of O2 uptake. These data are consistent with an O2-requiring oxidation of the prosthetic group of acyl-CoA dehydrogenase to yield H2O2, which is broken down by catalase in the glyoxysome. Acetyl-CoA produced by β oxidation is consumed in the glyoxylate cycle, which is also located in this organelle.

The ATP:urea amido-lyase reaction of yeast has been shown to consist of two distinct activities, an avidin-sensitive, thiourea-insensitive urea carboxylase and an avidin-insensitive, thiourea-sensitive allophanate hydrolase. Mutants lacking each of these activities have been isolated and were found to complement each other.

The recent isolation of the glyoxysome, a cellular organelle containing the enzymes of the glyoxylate cycle, raised the possibility that this metabolic sequence may be compartmentalized in plant tissues actively converting fat to carbohydrate. To sustain this view it is necessary to demonstrate that all of the pertinent enzymes are present in quantities consistent with the conversion of fat to sucrose in the intact tissue. To this end we have assayed each of the enzymes of the citric acid and glyoxylate cycles in preparations of mitochondria and glyoxysomes from 5-day-old castor bean endosperm. This survey shows that the glyoxysome contains more than 85% of the total isocitratase and malate synthetase activities, and the mitochondria less than 5%. With the exception of aconitase, which appears to be easily solubilized, all enzymatic activities observed in the glyoxysome are sufficient to support rates of acetate metabolism that are greater than those known to be occurring in the intact tissue. In addition to those enzymes which have been previously reported to be compartmented in the glyoxysome, a specific glutamate: oxalacetate transaminase has been found in very high concentrations in both the glyoxysomes and the mitochondria. Our inability to detect succinic dehydrogenase and fumarase in the glyoxysome suggests that the succinate produced from isocitrate cleavage must be transported to the mitochondria (where these activities are high) before undergoing oxidation to malate. With the use of isolated particle preparations, isotopic evidence supporting this conclusion has been obtained.

Using specifically labeled 3-3H-phosphoenolpyruvate, the stereochemistry of CO2 (or HCO3-) addition was determined for the reactions catalyzed by P-enolpyruvate carboxylase (from peanuts and Acetobacter xylinum), P-enolpyruvate carboxykinase (from pigeon liver), and P-enolpyruvate carboxytransphosphorylase (from Propionibacterium shermanii). In all cases the addition occurs from the same side of the plane of enzyme-bound P-enolpyruvate, the si side. These results relate the stereochemical course of the protons in these carboxylation reactions to that in the enolase, fumarase, and phosphoglucose isomerase reactions. Furthermore, the conservation of the addition in evolution implies that this stereochemistry may have significance to the reaction mechanism.

Urea carboxylase, an avidin-sensitive enzyme catalyzing the ATP-dependent carboxylation of urea, has been shown to catalyze this reaction in a sequence of at least two steps. In the presence of all of the required carboxylation cofactors except urea, an enzyme-CO2 complex is formed. This complex was isolated by Sephadex chromatography and was capable, in the absence of added cofactors, of transferring the bound CO2 to urea. In addition to the carboxylation of urea this protein was capable of carboxylating free d-biotin, as well as the urea analogues formamide, formylurea, and acetamide. These data are consistent with the suggestion that urea carboxylase follows a reaction mechanism similar to those of the more thoroughly studied propionyl-CoA, acetyl-CoA, and pyruvate carboxylases.

We have previously determined the amounts of time required to complete various macromolecular synthetic processes needed for induction of allophanate hydrolase in Saccharomyces cerevisiae. This information provided a means of testing, in vivo, an early hypothesis suggesting that cycloheximide inhibited the initiation as well as elongation steps of protein synthesis. Our data suggest that initiation of protein synthesis in yeast may be inhibited by low concentrations of cycloheximide which do not significantly affect polypeptide chain elongation.

The active species of "CO2," i.e. CO2 or HCO3-, utilized in the pyruvate carboxylase and phosphoenolypruvate carboxylase reaction have been determined with an assay predicated upon the fact that the hydration of CO2 is a kinetically slow and measurable reaction. The results of these assays in both cases are in reasonable agreement with those expected if HCO3- is assumed to be the functional species utilized by both enzymes.

The active species of "CO2", i.e. CO2 or HCO3-, utilized in the ribulose diphosphate carboxylase reaction was determined with the use of homogeneous preparations of the spinach enzyme. Employing an assay system predicated upon the observation that the hydration of CO2 requires more than 60 sec to reach equilibrium at temperatures below 15°, data were obtained which are quite compatible with those expected if CO2 is the active species.