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The gene of the proteinase yscA inhibitor I A3, PAI3, of the yeast Saccharamyces cerevisiaewas isolated by oligonucleotide screening of a genomic DNA library and sequenced. The gene codes for a single protein of 68 amino acids. The structural PAI3gene was deleted in vitro by oligonucleotide-site-directed mutagenesis. The mutated allele was introduced via homologous recombination into the genome of wild-type yeast and into the genome of a yeast mutant, which lacks the second cytoplasmic proteinase-inhibitor, I B2. The deficiency of either or of both inhibitors has no effect on the cell viability under various physiological conditions. The inhibitor mutants, however, show an increase in the general in vivo protein degradation rate. The I A3mutant has a 2–3-fold increased protein degradation rate in the first 6 h after a shift from rich medium onto starvation-medium, whereas the I B2mutant shows a constantly increased degradation rate of 20–50% under the same conditions. The inhibitor double null mutant has the same protein degradation rate as the I A3null mutant. These results suggest an in vivo interaction between the vacuolor endopeptidases and their cytoplasmic inhibitors.

A new member of the endothelin/sarafotoxin family of vasoconstrictor peptides, bibrotoxin (BTX), was isolated from the venom of the burrowing aspAtractaspis bibroniby reversed‐phase FPLC. The amino acid sequence of BTX differs from SRTX‐b in the substitution Ala4instead of Lys4, which suggests that it represents the peptide isoform of Atractaspis bibronicorresponding to SRTX‐b. BTX competed for [125I]ET‐1 binding to human ETB‐type receptor with a Kiof 3.2 × 10−9M compared to 4.2 × 10−9M for SRTX‐b. In rat thorax aorta BTX induced vasoconstrictions with a threshold concentration of 3 × 10−8M compared to 1 × 10−9for ET‐1.

Localization in vacuoles, the lysosome‐like organelle of yeast, was checked for several newly detected proteolytic enzymes. While aminopeptidase Co and carboxypeptidase S were found in vacuoles, proteinase D and proteinase E as well as a variety of other proteolytic activities detectable with the aid of chromogenic peptide substrates do not reside in this cell compartment.

Carboxy-terminal processing of the mating pheromone α-factor of the yeast Saccharomyces cerevisiaehas been assumed to be due to the action of carboxypeptidase yscα. [(1985) EMBO J. 4, 173–177]. Here it is shown that a mutant ( kex1) defective in α-factor maturation is defective in carboxypeptidase yscα activity, indicating that the enzyme is indeed the processing catalyst. It is proposed that carboxypeptidase yscα is the product of the KEX1gene.

The KEX2gene-encoded, membrane-bound Ca 2+-dependent thiol endoproteinase, proteinase yscF, responsible for processing of the precursor protein of the sex pheromone α-factor of the yeast Saccharomyces cerevisiaewas solubilized from the membraneous fraction and partially purified. Gel filtration revealed an apparent M rof the native protein of around 150 000. Ca 2+concentration for half-maximal activity was in the micromolar range and concentration of the substrate Cbz-Tyr-Lys-Arg-4-nitroanilide for half-maximal velocity was 0.05 mM. The enzyme is able to cleave basic amino acids from the carboxy-terminus of peptides and probably involved in final maturation of the α-factor peptides generated by proteinase yscF is membrane-associated, active at neutral pH and responds strongly to the serine proteinase inhibitor phenylmethylsulfonyl fluoride as well as to -SH group blocking agents.

An O 2-consuming side reaction of d-ribulose 1,5-bisphosphate carboxylase causes photorespiration in plants. This reaction may be an inevitable consequence of the enzyme's inability to protect its ene-diolate reaction intermediate from O 2, a notion that is supported by the failure of persistent efforts to eliminate selectively its oxygenase activity by genetic manipulation. We have examined two aldolases with similar ene-diolate intermediates, l-rhamnulose 1-phosphate aldolase and l-fuculose 1-phosphate aldolase. The former enzyme has an oxygenase activity, while the latter does not, suggesting that the reaction with O 2is not inevitable.

Experiments were carried out to provide direct evidence that pyrophosphate: fructose-6-phosphate phosphotransferase (PFP) can operate as a glycolytic enzyme in some circumstances. A large increase in the rate of glycolysis was produced by adding uncoupler and alkalinising the medium of heterotrophic Chenopodium rubrumcells. Initially, a marked decrease in phospho enolpyruvate, 3-phosphoglycerate and hexose phosphates occurred, but no change in fructose 2,6-bisphosphate or inorganic pyrophosphate was observed. However, a gradual increase in the rate of O 2uptake during the subsequent 5 min was accompanied by an increase in fructose 2,6-bisphosphate and a decrease in pyrophosphate, providing evidence that activation of PFP contributes to the increase in rate of glycolysis. This was accompanied by partial recovery of the adenine nucleotide energy status.

Proteinase yscE of the yeast Saccharomyces cerevisiaehas been compared with the 20 S cylinder particles of Xenopuslaevis. Both proteins are characterized by a similar group of 10–12 polypeptides with molecular masses ranging between 21 and 38 kDa. Antibodies generated against the 20 S Xenopuscylinder particles show cross-reactivity with yeast proteinase yscE subunits. The Xenopusparticles and yeast proteinase yscE exhibit an identical image in electron microscopy. Both proteins appear as hollow cylinders mostly composed of four stacked annuli. The Xenopus20 S particles exhibit proteolytic activity against the three peptide derivatives known to be substrates of proteinase yscE. The pH optimum for activity and the inhibition spectrum of the proteolytic activities of Xenopus20 S particles and of yeast proteinase yscE are identical. The RNA content of the cylinder particles and of proteinase yscE is below 0.1 RNA chain per molecule. Our data suggest that proteinase yscE from yeast and the 20 S cylinder particles of X. laevisare homologous, highly conserved proteins carrying the catalytic character of a peptidase.

Proteasomes are large multicatalytic proteinase complexes found in all eukaryotic organisms investigated so far. They have been shown to play a central role in cytosolic and nuclear proteolysis. According to their sedimentation coefficients two types of these particles can be distinguished: 20S proteasomes and 26S proteasomes. In contrast to 20S proteasomes, which were mainly characterized on the basis of their ability to cleave small chromogenic peptide substrates and certain proteins in an ATP-independent manner, 26S proteasomes degrade ubiquitinylated proteins in an ATP-dependent reaction. 20S proteasomes have been found in all eukaryotes from yeast to man. So far 26S proteasomes have only been discovered in higher eukaryotes. We now report the existence of the 26S proteasome in a lower eukaryote, the yeast Saccharomyces cerevisiae. Formation of the 26S proteasome could most effectively be induced in crude extracts of heat stressed yeast cells by incubation with ATP and Mg 2+ions. This treatment yielded a protein complex, which eluted from gel filtration columns at molecular masses higher than 1500 kDa. Besides chromogenic peptide substrates, this complex cleaves ubiquitinylated proteins in an ATP-dependent fashion. In non-denaturing-PAGE, the purified 26S proteasome disintegrated and migrated as four protein bands. One of these bands could be identified as the 20S proteasome. On SDS-PAGE, the 26S proteasome showed a complex pattern of subunit bands with molecular masses between 15 and 100 kDa. Further evidence for the 20S proteasome being the proteolytically active core of the 26S proteasome was obtained by following peptide cleaving activities in extracts of yeast strains carrying mutations in various subunits of the 20S proteasome.

20S proteasomes are multifunctional proteinase complexes ubiquitous in eucaryotes. We have cloned the yeast PRE3gene by complementation of the pre3‐2mutation, which leads to a defect in the peptidylglutamyl‐peptide hydrolyzing activity of the 20S proteasome. The PRE3gene, a β‐type member of the proteasomal gene family, is essential for cellular life and codes for a 193‐amino acid proteasomal subunit with a predicted molecular mass of 21.2 kDa. The Pre3 protein shows striking homology to the human proteasome subunits Hsδ and Lmp2 (Ring12). Lmp2 is encoded in the major histocompatibility complex class II region implicating proteasomes in antigen processing.

Radioactively labelled material from yeast cells grown in the presence of [ 32P]phosphate was specifically recognized by antibodies raised against yeast phosphofructokinase. Purified yeast phosphofructokinase was phosphorylated in a cyclic AMP-independent manner by a protein kinase enriched from yeast extracts. This phosphorylation occurred specifically on the β-subunit, and 0.56 mol of phosphate/mol of subunit was incorporated. The results indicate the phosphorylation of yeast phosphofructokinase both in vivo and in vitro. Phosphofructokinase phosphorylated in vitro was more stable against proteolytic degradation compared to the non-phosphorylated enzyme.