Changes in ribosomal properties during adenylate deprivation in the cells of Kluyveromyces lactis

In an adenine-requiring mutant strain of the yeast, Kluyveromyces lactis, the intracellular content of ATP is one-third to one-fifth that in a prototrophic wild strain under growing conditions. The quantitative differences becomes rather small in resting stationary-phase cells. Temporary changes in the two-dimensional protein patterns of mutant ribosomes occur when the ATP content is lowest during the transition phase of growth. The transfer of exponentially growing cells to a synthetic complete medium void of adenine induces the same changes in mutant ribosomes within several hours. Identification of ribosomal proteins by two-dimensional gel electrophoresis indicated all changeable proteins (at least five proteins) to belong to 40S ribosomal subunits. The mutant ribosomes prepared from the transition-phase cells have much lower activity (below 60%) for poly(U)-directed polyphenylalanine synthesis than those in exponentially growing or resting stationary-phase cells. Thus, changes in ribosomal components associated with the differences in ribosome activity in a cell-free system were noted in the adenylate-deprived cells of K. lactis.     

II. Yeast sequencing reports. The sequence of 12·5 kb from the right arm of chromosome II predicts a new N-terminal sequence for the IRA1 protein and reveals two new genes,one of which is a DEAD-box helicase

We have determined the complete nucleotide sequence of a 12·5 kb segment from the right arm of chromosome II carried by the cosmid α20. The sequence encodes the 5′ end of the IRA1 gene. Two complete new open reading frames and the 3′ non-coding region of the SUP1 (SUP45) gene. A comparison of our sequence with the data bank reveals a 154 amino acid extension at the N-terminus of Ira1p compared to the previously predicted sequence. According to the 11th edition of the Saccharomyces cerevisiae genetic map, our sequence should encode the MAK5 gene, which is necessary for the maintenance of dsRNA killer plasmids. One of the two new open reading frames, YBR1119, is predicted to encode an RNA helicase, thus YBR1119 may correspond to the MAK5 gene. The sequence has been deposited in the EMBL data library under Accession Number X78937.     

Yeast flocculation: Lectin synthesis and activation

Yeast flocculation involves binding of surface lectins to carbohydrate receptors on neighbouring cell walls. Brewing strains of Saccharomyces cerevisiae normally become flocculent in the stationary phase of growth. This paper presents evidence that lectins are synthesized in exponential phase, inserted into the cell wall, and activated later at the time of flocculation onset. Cycloheximide failed to prevent flocculation unless it was added in early growth; with later additions progressively larger degrees of flocculation occurred. Flocculation onset was delayed by cycloheximide but was otherwise cycloheximide insensitive. Preflocculent cells could be artificially activated to full flocculation by heat. Artificial activation of samples from growing yeast cultures confirmed the progressive synthesis of lectins throughout exponential growth. Pronase E treatment of whole cells prior to heating prevented any activation of flocculation. It was concluded that lectins were synthesized continuously from an early stage of growth and rapidly inserted into the cell wall (accessible by pronase E), where they remained inactive for up to 14 h, before being activated at flocculation onset by an as-yet unknown mechanism. It was found that lectin synthesis and activation occurred in all brewing strains tested.     

A 12·8 kb segment,on the right arm of chromosome II from Saccharomyces cerevisiae including part of the DUR1, 2 gene,contains five putative new genes

A 12 820 bp fragment from the right arm of chromosome II of Saccharomyces cerevisiae was sequenced and analysed. This fragment contains six non-overlapping long open reading frames (ORFs) designated from the centromere- to the telomere-proximal ends as: YBR1441, 1443, 1444, 1445, 1446 and 1448. YBR1441 encodes a polypeptide of 845 amino acids which shares a long consensus domain with products of S. cerevisiae MCM2, MCM3, CDC46 and Schizosaccharomyces pombe cdc21⁺ genes. These genes are involved in DNA replication. YBR1445 encodes a polypeptide of 404 amino acids which has strong similarity with the S. cerevisiae KRE2/MNT1, YUR1, KTR1 gene products. The KRE2/MNT1 protein is an α-1,2-mannosyltransferase. The product of YBR1444, which encodes a protein of 375 amino acids, presents a lipase signature sequence and a peroxisomal targeting signal. YBR1448, whose sequence extends further on the telomere-proximal end of the fragment, is identical to the 3′ end of the DUR1,2 gene encoding urea amidolyase. The two ORFs, YBR1443 and YBR1446, exhibit no significant similarity with any known gene.     

Activation of trehalase during growth induction by nitrogen sources in the yeast Saccharomyces cerevisiae depends on the free catalytic subunits of camp-dependent protein kinase,but not on functional ras proteins

Addition of a nitrogen-source to glucose-repressed, nitrogen-starved G0 cells of the yeast Saccharomyces cerevisiae in the presence of a fermentable carbon source induces growth and causes within a few minutes a five-fold, protein-synthesis-independent increase in the activity of trehalase. Nitrogen-activated trehalase could be deactivated in vitro by alkaline phosphatase treatment, supporting the idea that the activation is triggered by phosphorylation. Yeast strains containing only one of the three TPK genes (which encode the catalytic subunit of cAMP-dependent protein kinase) showed different degrees of nitrogen-induced trehalase activation. The order of effectiveness was different from that previously reported for glucose-induced activation of trehalase in glucose-derepressed yeast cells. Further reduction of TPK-encoded catalytic subunit activity by partially inactivating point mutations in the remaining TPK gene further diminished nitrogen-induced trehalase activation, while deletion of the BCY1 gene (which encodes the regulatory subunit) in the same strains resulted in an increase in the extent of activation. Deletion of the RAS genes in such a tpk^w1 bcy1 strain had no effect. These results are consistent with mediation of nitrogen-induced trehalase activation by the free catalytic subunits alone. They support our previous conclusion that cAMP does not act as second messenger in this nitrogen-induced activation process and our suggestion that a novel nitrogen-induced signaling pathway integrates with the cAMP pathway at the level of the free catalytic subunits of protein kinase A. Western blot experiments showed that the differences in the extent of trehalase activation were not due to differences in trehalase expression. On the other hand, we cannot completely exclude that protein kinase A influences the nitrogen-induced activation mechanism itself rather than acting directly on trehalase. However, any such alternative explanation requires the existence of an additional, yet unknown, mechanism for activation of trehalase besides the well-established regulation by protein kinase A.     

Isolation and characterization of peroxisomal protein import (Pim−) mutants of Hansenula polymorpha

In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim^? phenotype). Genetic analysis of back-crossed mutants revealed five different complementation groups, which were designated PERI–PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim^? phenotypes were determined by single recessive nuclear mutations. The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome-borne enzymes in wild-type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild-type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.     

ASSIMILABLE NITROGEN ADDITION AND HEXOSE TRANSPORT SYSTEM ACTIVITY DURING ENOLOGICAL FERMENTATION

Addition of ammoniacal nitrogen to nitrogen-limited musts is widely used by enologists to reduce the time required for the completion of fermentation. Nevertheless the physiological basis of the actual effect of such an addition is not fully understood. The effect of ammoniacal addition was studied on a nitrogen-limited must under conditions whereby addition had no effect on yeast growth. In these conditions nitrogen addition promotes increase of protein synthesis rate and activity of the hexose transport system. The results suggest that the reactivation of the hexose transport system does not require a de novo biosynthesis of sugar carriers, but the synthesis of an unknown regulatory protein.     

Saccharomyces cerevisiae mata mutant cells defective in pointed projection formation in response to α-factor at high concentrations

We have isolated Saccharomyces cerevisiae MAT a mutant cells that do not form a pointed projection but elongate in response to α-factor at high concentrations. Complementation tests defined three genes, PPF1, PPF2, and PPF3 (for pointed projection formation), necessary for pointed projection formation. Allelism tests with genes known to be needed for projection formation revealed that PPF1 is identical to SPA2, while PPF2 and PPF3 are not allelic to SST2, STE2, SPA2, BEM1 or SLK1/SSP31/BCK1. The morphology of MAT a ppf mutants treated with high concentrations of α-factor is similar to that of MAT a PPF cells treated with α-factor at low concentrations. Quantitative mating tests showed that PPF2 and PPF3 are not essential for mating in either MAT a or MATα background. Monitoring of division arrest and expression of an α-factor-inducible gene revealed that mutations in the PPF genes do not affect the responses of MAT a cells to low concentrations of α-factor. Unlike wild-type cells, the ppf mutants exhibited early recovery from α-factor-induced division arrest. Furthermore, vegetatively growing ppf3-1 cells are slightly defective in cell separation of mother and daughter cells and in selection of the correct bud sites in all cell types. These results indicate that PPF2 and PPF3 are involved in the response to α-factor at high concentrations and that PPF3 is also required for proper establishment of polarity in vegetative growth.     

Physiological approach to heterologous human serum albumin production by Kluyveromyces lactis in chemostat culture

Production of recombinant human serum albumin (rHSA) controlled by the constitutive promoter phosphoglycerate kinase was studied in Kluyveromyces lactis. It was governed by both cell concentration and glycolytic flow. The triggering of the fermentation metabolism by unfavourable culture conditions (pH, pO₂, D) caused a decrease in the synthesis of the heterologous protein. The highest productivity (75 mg 1^?1 per h) and rHSA concentration (62 mg 1^?1) were obtained in chemostat culture with a dilution rate of 0·12 h^?1 and with 38 g 1^?1 dry weight.