The use of phenylmethylsulfonyl fluoride in the study of catabolite inactivation and repression in intact cells of Saccharomyces cerevisiae

Catabolite inactivation of fructose-1,6-bisphosphatase, isocitrate lyase, phosphoenolpruvate carboxykinase and malate dehydrogenase in intact cells could be prevented by phenylmethylsulfonyl fluoride added 40 min prior to the addition of glucose. Protein synthesis, fermentative and respiratory activity and catabolite repression were not affected. Elimination of catabolite inactivation by the addition of PMSF revealed that catabolite repression started at different times for different enzyme.Abbreviation PMSF phenylmethylsulfonyl fluoride     

Effect of glucose on isocitrate lyase in Phycomyces blakesleeanus.

Repression of the synthesis of isocitrate lyase by glucose and/or induction of the synthesis of isocitrate lyase by acetate in Phycomyces blakesleeanus were demonstrated. Both glycerol and ethanol failed to induce isocitrate lyase activity. Furthermore, glucose appeared to cause an in vivo catabolite inactivation of the derepressed enzyme. Isocitrate lyase was inactivated both reversibly and irreversibly by glucose.     

Degradation and turnover of peroxisomes in the yeast Hansenula polymorpha induced by selective inactivation of peroxisomal enzymes

Inactivation of peroxisomal enzymes in the yeast Hansenula polymorpha was studied following transfer of cells into cultivation media in which their activity was no longer required for growth. After transfer of methanol-grown cells into media containing glucose - a substrate that fully represses alcohol oxidase synthesis - the rapid inactivation of alcohol oxidase and catalase was paralleled by a disappearance of alcohol oxidase and catalase protein. The rate and extent of this inactivation was dependent upon conditions of cultivation of cells prior to their transfer. This carbon catabolite inactivation of alcohol oxidase was paralleled by degradation of peroxisomes which occurred by means of an autophagic process that was initiated by the formation of a number of electron-dense membranes around the organelles to be degraded. Sequestration was confined to peroxisomes; other cell-components such as ribosomes were absent in the sequestered cell compartment. Also, cytochemically, hydrolytic enzymes could not be demonstrated in these autophagosomes. The vacuole played a major role in the subsequent peroxisomal breakdown since it provided the enzymes required for proteolysis. Two basically similar mechanisms were observed with respect to the administration of vacuolar enzymes into the sequestered cell compartment. The first mechanism involved incorporation of a small vacuolar vesicle into the sequestered cell compartment. The delimiting membrane of this vacuolar vesicle subsequently disrupted, thereby exposing the contents of the sequestered cell compartment to vacuolar hydrolases which then degraded the peroxisomal proteins. The second mechanism, observed in cells which already contained one or more autophagic vacuoles, included fusion of the delimiting membranes of an autophagosome with the membrane surrounding an autophagic vacuole which led to migration of the peroxisome inside the latter organelle. Peroxisomes of methanol-grown H. polymorpha were degraded individually. In one cell 2 or 3 peroxisomes might be subject to degradation at the same time, but they were never observed together in one autophagosome. However, fusions of autophagic vacuoles in one cell were frequently observed. After inhibition of the cell's energy-metabolism by cyanide ions or during anaerobic incubations the formation of autophagosomes was prevented and degradation was not observed.     

Catabolite inactivation of isocitrate lyase from Saccharomyces cerevisiae

A reversible carbon catabolite inactivation step is described for isocitrate lyase from Saccharomyces cerevisiae. This reversible inactivation step of isocitrate lyase is similar to that described for fructose 1,6-bisphosphatase. Addition of 2,4-dinitrophenol, nystatin or glucose to cultures, grown in ethanol as carbon source, caused a rapid loss of the isocitrate lyase and fructose 1,6-bisphosphatase activities at pH 5.5 but not at pH 7.5. These results suggest that intracellular acidification and thus a cAMP increase is involved in the catabolite inactivation mechanism of both enzymes. From results obtained by addition of glucose to yeast cultures at pH 7.5 it was concluded that others factors than cAMP can play a role in the catabolite inactivation mechanism of both enzymes.     

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans

     The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creA ^d -30 strain showed that the creA gene is not involved in this process. Received: 13 May 1994 / Accepted 12 August 1994     

Peroxisomal isocitrate lyase of the n-alkane-assimilating yeast Candida tropicalis: gene analysis and characterization

A genomic DNA clone encoding isocitrate lyase, a key enzyme of the glyoxylate cycle and a peroxisomal enzyme of the n-alkane-assimilating yeast Candida tropicalis has been isolated with a cDNA probe from the yeast lambda EMBL library. Nucleotide sequence analysis of the genomic DNA clone disclosed that the region coding isocitrate lyase had a length of 1,650 base pairs, corresponding to 550 amino acids (61,602 Da). RNA blot analysis demonstrated that only one kind of mRNA (2 kb) supposed to be transcribed from this gene was present in the cells. A comparison of the amino acid sequences was made with the isocitrate lyase of castor bean, one of the glyoxysomal enzymes, and the enzyme of E. coli. The isocitrate lyases of C. tropicalis and castor bean had high homology, and the presence of some amino acid stretches conserved in all three enzymes suggests that these might be involved in the catalysis of the common reaction. There was an insertion common to the isocitrate lyases of C. tropicalis and castor bean, which is of interest concerning their evolution. In the C-terminal region, a characteristic sequence similar to that previously proposed as the import signal to peroxisomes was present.     

Control of isocitrate lyase in Nocardia salmonicolor (NCIB9701).

Nocardia salmonicolor, grown on acetate, commercial D,L-lactate or hydrocarbon substrates, has high isocitrate lyase activities compared with those resulting from growth on other carbon sources. This presumably reflects the anaplerotic role of the glyoxylate cycle during growth on the former substrates. Amongst a variety of compounds tested, including glucose, pyruvate and tricarboxylic acid cycle intermediates, only succinate and fumarate prevented an increase in enzyme activity in the presence of acetate. When acetate (equimolar to the initial sugar concentration) was added to cultures growing on glucose, there followed de novo synthesis of isocitrated lyase and isocitrate dehydrogenase, with increases in growth rate and glucose utilization, and both acetate and glucose were metabolized simultaneously. A minute amount of acetate (40 muM) caused isocitrate lyase synthesis (a three-fold increase in activity within 3 min of addition) when added to glucose-limited continuous cultures, but even large amounts added to nitrogen-limited batch cultures were ineffective. Malonate, at a concentration that was not totally growth-inhibitory (1mM) prevented the inhibition of acetate-stimulated isocitrate lyase synthesis by succinate, but fumarate still inhibited in the presence of malonate. Phosphoenolpyruvate is a non-competitive inhibitor of the enzyme (apparent Ki 1-7 mM). The results are consistent with the induction of isocitrate or a closely related metabolite, and catabolite repression by a C-4 acid of the tricarboxylic acid cycle, possibly fumarate.     

Constitutive appearance of peroxisomes in a regulatory mutant of the methylotrophic yeast Hansenula polymorpha

Summary A selection by glucosamine for mutants of Hansenula polymorpha insensitive to glucose repression of methanol assimilation is described. Constitutive synthesis of enzymes is established in standard batch cultures of glucosegrown cells. Upon prolonged glucose metabolism the phenotype is masked by catabolite inactivation and degradation of enzymes. Addition of the substrate methanol remarkably improves constitutive synthesis by preventing catabolite inactivation and delaying degradation. Regular peroxisomes of reduced number are formed in mutant cells under repressed conditions. No constitutive synthesis is detectable using ethanol as a carbon source. In addition, this alcohol is detrimental to growth of the mutants, indicating that H. polymorpha is constrained to repress synthesis of enzymes involved in the C₁-metabolism when ethanol is present as a substrate.     

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes in Arabidopsis Hypocotyls during Seedling Growth

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.     

Production of cell wall-degrading enzymes by Aspergillus nidulans: a model system for fungal pathogenesis of plants.

The cell wall-degrading enzymes polygalacturonase and pectate lyase have been suggested to be crucial for penetration and colonization of plant tissues by some fungal pathogens. We have found that Aspergillus nidulans (= Emericella nidulans), a saprophytic Ascomycete, produces levels of these enzymes equal to those produced by soft-rotting Erwinia species. Induction of polygacturonase and pectate lyase in A. nidulans requires substrate and is completely repressed by glucose. Surprisingly, inoculation of excised plant tissues with A. nidulans conidia leads to formation of necrotic, water-soaked lesions within which the organism sporulates. Thus, A. nidulans has phytopathogenic potential. The release of glucose and other sugars from wounded tissues may repress pectolytic enzyme production and limit disease development. Therefore, we tested creA204, a mutation that relieves glucose repression of some A. nidulans carbon utilization enzymes, for its effect on production of pectolytic enzymes. creA204 failed to relieve catabolite repression of polygalacturonase or pectate lyase and had no effect on disease severity.     

Cyclic AMP,fructose-2,6-bisphosphate and catabolite inactivation of enzymes in the hydrocarbon-assimilating yeast Candida maltosa

The inactivation of fructose-1,6-bisphosphatase, isocitrate lyase and cytoplasmic malate dehydrogenase in Candida maltosa was found to occur after the addition of glucose to starved cells. The concentration of cyclic AMP and fructose-2,6-bisphosphate increased drastically within 30 s when glucose was added to the intact cells of this yeast. From these results it was concluded that catabolite inactivation, with participation of cyclic AMP and fructose-2,6-bisphosphate, is an important control mechanism of the gluconeogenetic sequence in the n-alkane-assimilating yeast Candida maltosa, as described for Saccharomyces cerevisiae.     

Pexophagy: the selective autophagy of peroxisomes

Pichia pastoris and Hansenula polymorpha are methylotrophic yeasts capable of utilizing methanol, as a sole source of carbon and energy. Growth of these yeast species on methanol requires the synthesis of cytosolic and peroxisomal enzymes combined with the proliferation of peroxisomes. Peroxisomes are also abundantly present in the alkane-utilizing yeast Yarrowia lipolytica upon growth of cells on oleic acid. This feature has made these yeast species attractive model systems to dissect the molecular mechanisms controlling peroxisome biogenesis. We have found that upon glucose- or ethanol-induced catabolite inactivation, metabolically superfluous peroxisomes are rapidly and selectively degraded within the vacuole by a process called pexophagy, the selective removal of peroxisomes by autophagy-like processes. Utilizing several genetic screens, we have identified a number of genes that are essential for pexophagy. In this review, we will summarize our current knowledge of the molecular events of pexophagy.     

Induction of β-oxidation enzymes and microbody proliferation in Aspergillus nidulans

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced β-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the β-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The β-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans. Received: 8 March 1996 / Accepted: 5 August 1996     

Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.     

Catabolite inactivation of the maltose transporter in nitrogen-starved yeast could be due to the stimulation of general protein turnover

Addition of glucose to Saccharomyces cerevisiae inactivates the maltose transporter. The general consensus is that this inactivation, called catabolite inactivation, is one of the control mechanisms developed by this organism to use glucose preferentially whenever it is available. Using nitrogen-starved cells (resting cells), it has been shown that glucose triggers endocytosis and degradation of the transporter in the vacuole. We now show that maltose itself triggers inactivation and degradation of its own transporter as efficiently as glucose. This fact, and the observation that glucose inactivates a variety of plasma membrane proteins including glucose transporters themselves, suggests that catabolite inactivation of the maltose transporter in nitrogen-starved cells is not a control mechanism specifically directed to ensure a preferential use of glucose. It is proposed that, in this metabolic condition, inactivation of the maltose transporter might be due to the stimulation of the general protein turnover that follows nitrogen starvation.     

Effect of glucose and cyclic adenosine 3',5'-monophosphate on the synthesis of succinate dehydrogenase and isocitrate lyase in Escherichia coli.

Repressed respiration of Escherichia coli cells grown in the presence of 2% glucose was derepressed when the cells were incubated in a buffer containing casamino acids. The glucose-repressed cells were deficient in succinate dehydrogenase [EC 1.3.99.1] and isocitrate lyase [EC 4.1.3.1] activities, which increased during the incubation. The increases in respiratory activity and enzyme activity on incubation were repressed by glucose, but except for isocitrate lyase these repressions could be restored by the addition of cyclic adenosine 3',5'-monophosphate. Inhibitors of protein synthesis blocked the increase of enzyme activity on incubation without glucose, or with glucose and the cyclic nucleotide.     

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway

Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.     

A hexose transporter homologue controls glucose repression in the methylotrophic yeast Hansenula polymorpha

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser(85)) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.