Biomedical subjects

W Bandlow

Publications and source records attributed to W Bandlow.

At least 19 recordsLinked to original sources

PubMed2001

Two parameters improve efficiency of mitochondrial uptake of adenylate kinase: decreased folding velocity and increased propensity of N-terminal alpha-helix formation.

The long isoform of eukaryotic adenylate kinase has a dual subcellular location in the cytoplasm and in the mitochondrial intermembrane space. Protein sequences and modifications are identical in both locations. In yeast, the bulk of the major form of adenylate kinase (Aky2p) is in the cytoplasm and, in the steady state, only 5-8% is sorted to the mitochondrial intermembrane space. Since the reasons for exclusion from mitochondrial import are unclear, we have constructed aky2 mutants with elevated mitochondrial uptake efficiency of Aky2p in vivo and in vitro. We have analyzed the effect of the mutations on secondary structure prediction in silico and have tested folding velocity and folding stability. One type of mutants displayed decreased proteolytic stability and retarded renaturation kinetics after chaotropic denaturation implying that deterioration of folding leads to prolonged presentation of target information to mitochondrial import receptors, thereby effecting improved uptake. In a second type of mutants, increased import efficiency was correlated with an increased probability of formation of an alpha-helix with increased amphipathic moment at the N-terminus suggesting that targeting interactions with mitochondrial import receptors had been improved at the level of binding affinity.

Adenylate Kinase↗

PubMed2000

Stable plasma membrane expression of the soluble domain of the human insulin receptor in yeast.

The soluble cytoplasmic kinase domain of the human insulin receptor was N-terminally equipped with either an N-acetylation or a dual-acylation motif (MGC box, to allow myristoylation/palmitoylation) and expressed in yeast cells under the control of the inducible CUP1 promoter. Although the cellular concentration was about the same in both instances (reflecting similar stability against proteolysis), only the myristoylated protein was capable of autophosphorylation to a significant extent and was active to phosphorylate endogenous yeast proteins at tyrosine residues in vivo. Cellular subfractionation showed that the insulin receptor was associated with plasma membranes, from where it was not extractable with high salt or alkali, but a significant fraction was also localized in the nuclear fraction. The myristoylated protein is absent from the cytoplasm. No effect of expression of either the acetylated or the myristoylated version on growth and respiration on various carbon sources was detected, suggesting a failure of the active insulin receptor kinase domain to couple to yeast (glucose) signalling cascades.

Blotting, Western↗

PubMed2000

Insulin-like signaling in yeast: modulation of protein phosphatase 2A, protein kinase A, cAMP-specific phosphodiesterase, and glycosyl-phosphatidylinositol-specific phospholipase C activities.

Previously, we have described significant effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae under conditions of growth limitation. These regulations apparently rely on a transmembrane receptor capable of binding human insulin and responding by tyrosine/serine phosphorylation of a specific set of polypeptides [Müller, G., Rouveyre, N., Crecelius, A., and Bandlow, W. (1998) Biochemistry 37, 8683-8695; Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W. (1998) Biochemistry 37, 8696-8704; Müller, G., Rouveyre, N., Upshon, C., and Bandlow, W. (1998) Biochemistry 37, 8705-8713]. To characterize the molecular link between the initial steps in insulin-like signaling in yeast and the changes in the activities of glycogen synthase and glycogen phosphorylase, we examined here the effects of human insulin on a set of key regulatory enzymes of glycogen metabolism, protein phosphatase 2A (PP2A), cAMP-specific phosphodiesterase (cAMP-PDE), and protein kinase A (PKA). PP2A was activated about 2-fold by insulin in spheroplasts and in intact cells, whereas the fraction of active PKA was significantly reduced in a cAMP-independent manner as well as through a subsequent up to 3-fold increase in particulate cAMP-PDE activity accompanied by a 50% decrease in cytosolic cAMP levels. In addition, glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC), which in isolated rat adipocytes is activated by insulin, was stimulated to up to 5-fold by glucose and 10-fold by glucose plus insulin in both yeast spheroplasts and intact cells leading to a concentration-dependent leftward shift of the glucose-response curve for activation of the GPI-PLC. GPI-PLC was most pronouncedly stimulated by authentic human insulin compared to various insulin analogues and insulin-like growth factor I. In addition to lipolytic cleavage by GPI-PLC, the GPI anchor of the cAMP-binding ectoprotein, Gce1p, was secondarily processed by a rapid proteolytic event. As the GPI-PLC reaction is rate limiting, the efficiency of the two-step anchor cleavage was significantly increased when insulin was present together with glucose as compared to glucose alone. The insulin concentrations effective in modulating PP2A, PKA, cAMP-PDE, and GPI-PLC activities correlate well with those required for half-saturation of the specific binding sites as well as for stimulation of protein phosphorylation and glycogen accumulation. The data suggest that mammalian insulin-sensitive cells and yeast share (part of) the key regulatory mechanism (consisting of PP2A, PKA, cAMP-PDE, and GPI-PLC) involved in the transduction of the insulin signal from the respective receptor systems to glycogen synthase and phosphorylase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

The yeast trimeric guanine nucleotide-binding protein alpha subunit, Gpa2p, controls the meiosis-specific kinase Ime2p activity in response to nutrients.

Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development. Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation. Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro. Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen. In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function. Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium. These observations are consistent with the following model. In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels. Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium. The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted. This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision.

Base Sequence↗

PubMed1998

Insulin signaling in the yeast Saccharomyces cerevisiae. 1. Stimulation of glucose metabolism and Snf1 kinase by human insulin.

Effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae were studied in this report. Under two conditions of growth limitation (glucose-grown cells during transition to stationary phase or spheroplasts during incubation in synthetic glucose medium), human insulin (10 and 1 microM, respectively) enhanced glycogen accumulation and glycogen synthase activity by 40-60% compared to control cells. Glycogen phosphorylase activity was also increased under the same conditions, but this stimulation was diminished by 35-45% in insulin-treated compared to control cells. Thus, under growth limitation, insulin causes glycogen phosphorylase and glycogen synthase to become more sensitive to inactivation and activation, respectively. In glucose-induced spheroplasts, insulin (1 microM), in addition to glycogen accumulation, led to about 2-fold increases of the rates of ethanol production and glucose oxidation compared to control cells, and the maximal concentration of hexose 6-phosphate was increased by 30-40%. In contrast, glucose transport as well as the levels of the allosteric regulators, fructose 2,6-bisphosphate and cAMP, were not altered at all. Snf1 kinase is assumed to be involved in the regulation of glycogen metabolism in yeast, although it does not seem to be modulated directly by the glucose concentration. Snf1 kinase activity was elevated 5-10-fold in response to insulin both during glucose induction of yeast spheroplasts and during transition to stationary phase of glucose-grown cells. We conclude that Saccharomyces cerevisiae and insulin-sensitive mammalian cells share some parts of the signaling cascades regulating oxidative and nonoxidative glucose metabolism in response to glucose and insulin.

Biological Transport↗

PubMed1998

Insulin signaling in the yeast Saccharomyces cerevisiae. 2. Interaction of human insulin with a putative binding protein.

A putative insulin-binding protein (Kd = 0.5 +/- 0.2 microM for human insulin) was partially purified from solubilized plasma membranes of Saccharomyces cerevisiaeby wheat germ agglutinin and insulin affinity chromatographies. The binding affinities of various mutant insulin analogues correlated well with their capacities to activate glycogen synthase and SNF1 kinase in glucose-induced yeast spheroplasts, the ranking of their relative efficacies in yeast and in isolated rat adipocytes being similar. Using a bifunctional cross-linker and two different experimental protocols, a 53-kDa polypeptide contained in the insulin-binding protein preparation was specifically affinity cross-linked to [125I]monoiodo[B26]insulin. The relative rankings of the insulin analogues with respect to inhibition of cross-linking and binding to the partially purified insulin-binding protein were identical. Incubation of intact yeast spheroplasts with [125I]monoiodo[AI4]insulin led to specific and time-dependent association of the radiolabeled insulin with the cell surface followed by its internalization and degradation. These processes were considerably delayed by low temperature and energy depletion of the spheroplasts, suggesting involvement of the ATP-dependent endosomal apparatus. These data provide evidence for the existence of a low-affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae.

Animals↗

PubMed1998

Insulin signaling in the yeast Saccharomyces cerevisiae. 3. Induction of protein phosphorylation by human insulin.

A low affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae has been identified recently (Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W., preceding paper in this issue). Since the mammalian insulin receptor functions as a tyrosine kinase with autophosphorylation capacity, kinase studies were performed with the partially purified insulin-binding protein preparation. Incubation with [gamma-32P]ATP in vitro led to phosphorylation of the 53-kDa insulin-binding protein on serine but not on tyrosine residues. In addition, a 70-kDa polypeptide, copurified with the insulin-binding protein preparation, was tyrosine-phosphorylated under the same conditions. Phosphorylation of both proteins was enhanced by human insulin. These results obtained by immunoprecipitation and immunoblotting using specific anti-phosphoserine/threonine/tyrosine antibodies were confirmed by phosphoamino acid analysis of the individual immunoprecipitated and gel-purified 32P-labeled phosphoproteins. During gel filtration, the 53-kDa protein coeluted as a 300-kDa complex together with the 70-kDa phosphotyrosine-containing protein and was coimmunoprecipitated with the latter using an anti-phosphotyrosine antibody, strongly arguing for complex formation between the two proteins. The data presented raise the possibility that stimulation of glycogen synthesis by insulin in yeast is mediated by a 53-kDa insulin-binding protein and a 70-kDa phosphotyrosine-containing protein which are organized in a large plasma membrane-bound signaling complex. Elucidation of the function and molecular mode of interaction of these components in yeast may help to understand metabolic insulin signaling in mammalian cells.

Fungal Proteins↗

PubMed1998

Influence of N-terminal sequence variation on the sorting of major adenylate kinase to the mitochondrial intermembrane space in yeast.

Major adenylate kinase (Aky2p) from yeast has no cleavable presequence and occurs in identical form in the mitochondrial intermembrane space (6-8%) and in the cytoplasm (approx. 90%). To identify the signal(s) on Aky2p that might be required for mitochondrial import, the N-terminal region was examined. The N-terminus of Aky2p can guide at least two cytoplasmic passengers, dihydrofolate reductase from mouse and UMP kinase (Ura6p) from yeast, to the intermembrane space in vivo, showing that the N-terminus harbours import information. In contrast, deletion of the eight N-terminal amino acid residues or the introduction of two compensating frameshifts into this segment does not abolish translocation into the organelle's intermembrane space. Thus internal targeting and sorting information must be present in Aky2p as well. Neither a pronounced amphiphilic alpha-helical moment nor positive charges in the N-terminal region is a necessary prerequisite for Aky2p to reach the intermembrane space. Even a surplus of negative charges in mutant N-termini does not impede basal import into the correct submitochondrial compartment. The potential to form an amphipathic alpha-helical structure of five to eight residues close to the N-terminus significantly improves import efficiency, whereas extension of this amphipathic structure, e.g. by replacing it with the homologous segment of Aky3p, a mitochondrial matrix protein from yeast, leads to misdirection of the chimaera to the matrix compartment. This shows that the topogenic N-terminal signal of Aky3p is dominant over the presumptive internal intermembrane space-targeting signal of Aky2p and argues that the sorting of wild-type Aky2p to the intermembrane space is not due to the presence in the protein of a specific sorting sequence for the intermembrane space, but rather is the consequence of being imported but not being sorted to the inner compartment. Some Aky2 mutant proteins are susceptible to proteolysis in the cytoplasm, indicating incorrect folding. They are nevertheless efficiently rescued by uptake into mitochondria, suggesting a negative correlation between folding velocity (or folding stability) and efficiency of import.

Adenylate Kinase↗

PubMed1998

Growth-regulated formation of heteromeric complexes of the centromere and promoter factor, Cbf1p, in yeast.

Transcriptional regulation of the yeast cytochrome c1 gene (CYT1) in response to oxygen and carbon source is mediated by Haplp and the Hap2 complex. Furthermore, the centromere-binding factor 1 (Cbflp) associates with the CYT1 upstream region (UAS(CYT1)), but its direct activation potential is insignificant. The possible role of Cbflp as a modulator of transcriptional adaptation to changes in nutritional conditions was examined. In electrophoretic mobility shift assays (EMSA) using yeast nuclear extracts, Cbflp was found to exist as homo- and heterodimers of processed subforms of 54 and 37 kDa. An additional 18-kDa version was the only species found in anaerobic cells grown under an atmosphere of purified nitrogen, but not when CO2 was used to establish anaerobiosis. All three dimers of the 37 and 54 kDa versions of Cbflp that occurred in oxidatively growing cells gave rise to hetero-oligomeric complexes containing other as yet unidentified protein(s). Complex formation was not observed with extracts from cultures grown on high levels of glucose and was dependent on pre-assembly in the absence of target DNA. Pre-treatment with alkaline phosphatase enhanced formation of these higher-order complexes. The C-terminal 18-kDa segment of Cbflp, which can undergo dimerization and bind DNA, does not induce supershifts after preincubation and is not influenced by dephosphorylation. We propose that the N-terminal domain is subject to carbon source- or growth-dependent phosphorylation/dephosphorylation events that result in differential recruitment of additional factors to promoters of genes that encode proteins required for non-fermentative growth.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed1997

The type of basal promoter determines the regulated or constitutive mode of transcription in the common control region of the yeast gene pair GCY1/RIO1.

The yeast genes, GCY1 and RIO1, are transcribed divergently from the 869-base pair intergenic region. GCY1 is inducible by galactose about 25-fold due to Gal4p-binding to a single UASGAL, whereas RIO1 is constitutively expressed. GCY1 has a TATA box obeying the consensus TATAAA, whereas the RIO1 5'-upstream region lacks such a motif. In vitro mutagenesis of the TATA motif of GCY1, on the one hand, and introduction of a TATA-element into the promoter of RIO1, on the other hand, as well as inversion of the intergenic region have revealed that transcription of GCY1 and RIO1 is only regulated by Gal4p when a consensus TATA motif is included in their core promoters but not in its absence. The data imply that only transcription complexes that assemble at a consensus TATA box are compatible with specific transactivators, such as Gal4p. As a result, the adjacent gene is subject to regulated expression. By contrast, if a consensus TATA sequence is absent, the initiation complex does not respond to regulatory transcription factors, and consequently, the respective gene is constitutively transcribed. On the other hand, we show that two blocks of homo-oligomeric (dA.dT) sequences do not function as boundary sequences that might confine regulatory action of Gal4p to GCY1.

Binding Sites↗

PubMed1997

Bleomycin hydrolase (Blh1p), a multi-sited thiol protease in search of a distinct physiological role.

Bleomycin hydrolase, Blh1p, from yeast was co-purified with Gce1p, a cAMP-binding ectoprotein, anchored to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Blh1p is a hydrophilic thiol protease lacking transmembrane domains. We have used polyclonal antibodies to study the topology of the over-expressed protein in yeast and have found that it is amphitropic. Part of Blh1p is associated with plasma membranes, and most of the rest occurs in the cytosol. Both the growth conditions and calcium were found to have minor influences on the topology of Blh1p, in that glucose and the earth-alkali ion slightly enhanced recruitment to the membrane. We have examined the possibility that co-purification of Blh1p with Gce1p has a functional basis, and have observed that over-expression of BLH1 in yeast leads to an acceleration of the glucose-induced amphiphilic to hydrophilic conversion of Gce1p, wherein Blh1p could either directly catalyse the proteolytic removal of the polar head-group of the GPI anchor subsequent to an initial lipolytic cleavage by a GPI-specific phospholipase C or indirectly modulate the reaction. The data show that a thiol protease is involved, but point to an indirect role of Blh1p in GPI processing. Proteases with similar or overlapping substrate specificity are likely to exist, since deletion of BLH1 neither entails a growth-defect on any carbon source tested, nor the loss of proteolytic processing of the GPI anchor of Gce1p. Reduced proteolytic GPI processing is, however, observed in the blh1 mutant and the corresponding acceleration in the respective BLH1 multi-copy transformant.

Calcium↗

PubMed1997

The general regulatory factor Reb1p controls basal, but not Gal4p-mediated, transcription of the GCY1 gene in yeast.

Expression of the gene GCY1 in Saccharomyces cerevisiae is induced by about 25-fold in the presence of galactose as a result of activation by Gal4p. In contrast to other Gal4p-regulated genes, such as GAL1 or GAL10, GCY1 is transcribed at a relatively high basal level. We have analysed the basis of this behaviour and have found that, in addition to a UASGAL, a binding site for the general regulatory factor Reb1p is localized 100 bp upstream of the TATA sequence and about 140 bp 3' to the UASGAL. Reb1p binds to this site with low affinity. Reb1p, an abundant, multifunctional DNA-binding protein in yeast, acts as a weak transcriptional activator in the control regions of several genes encoding unrelated functions. The action of Reb1p is assumed to be strongly position dependent. In the control region of GCY1. Reb1p acts independently of position and stimulates basal expression of GCY1 about threefold, whereas Gal4p-mediated activation is not influenced significantly. Promoter-proximal insertion of an additional Reb1p recognition site enhances basal transcription only marginally, but can largely compensate for deletion of the natural Reb1p-binding site. Either an Abf1p- or a Rap1p-binding site can substitute for the Reb1p recognition sequence, indicating that these general regulatory factors fulfill related functions in basal transcription, without affecting Gal4p-mediated activation. In addition to Reb1p, the sequence of the Gal4p-binding site influences basal transcription. This effect is independent of the Gal4 protein, as it operates in a gal4 mutant background as well. This finding suggests that the nucleotide sequence of the UASGAL in the GCY1 promoter has intrinsic properties, presumably a particular DNA structure, that influence basal transcription and act synergistically with Reb1p.

Binding Sites↗

PubMed1996

Interactions of the yeast centromere and promoter factor, Cpf1p, with the cytochrome c1 upstream region and functional implications on regulated gene expression.

The upstream activation site (UAS) of the cytochrome c1 gene, CYT1, contains sequences for DNA-binding of several transcription factors. Among them are the heme-dependent protein, Hap1p, and the multiprotein complex, Hap2/3/4/5, which mediate transcriptional induction under aerobic conditions and after exhaustion of glucose, respectively. The multiple interactions of nuclear proteins with the UAS region of CYT1 observed in electrophoretic mobility shift experiments are influenced by carbon source and oxygen tension, but are independent of both regulators, Hap1p and Hap2/3/4/5. All protein-DNA complexes obtained are solely due to the association of the centromere and promoter factor 1 (Cpf1p) with the centromere determining element (CDE I)-like motif at the 5' boundary of the UAS(CYT1). This motif overlaps with a consensus sequence for the binding of the general factor Abf1p. Functional analyses after the separate introduction of point mutations into both elements reveal no role for the latter protein and only a minor role for Cpf1p in the regulated expression of CYT1/lacZ chimaeric proteins. However, in cpf1-mutants, induction of CYT1 reaches higher steady state levels and adaptation to aerobic conditions occurs faster than in wild-type. Thus, Cpf1p seems to reduce CYT1 promoter activity under partly inducing conditions, e.g. when only one of the activators, Hap1p or the Hap2 complex, exerts its function.

Base Sequence↗

PubMed1996

Cytoplasmic and mitochondrial forms of yeast adenylate kinase 2 are N-acetylated.

Yeast major adenylate kinase (Aky2p), encoded by a single gene, occurs in two subcellular compartments, mitochondria and cytoplasm. Only 6-8% of the protein which has no cleavable presequence is imported into the organelle (Bandlow et al. (1988) Eur. J. Biochem. 178, 451-457). In the wild type two AKY2-derived signals (a major and a minor one) were detected by a monospecific antibody after two-dimensional gel electrophoresis and Western blotting. The signals reflected identical electrophoretic mobilities and were absent from an AKY2-disrupted strain suggesting that they were due to differently modified forms of Aky2p. Two similar signals were found in a mutant defective in protein N-acetylation, however, the pI values of both spots were shifted towards alkaline pH by one charge. This indicated that both forms of Aky2p were N-acetylated in the wild type and that their charge difference was not caused by incomplete N-acetylation. This observation furthermore suggested that, in the wild type, two different modifications exist one of which is N-acetylation. The second modification remains unidentified. We analysed the influence of protein N-acetylation on mitochondrial import. Both versions of Aky2p occurred in the cytoplasm and in mitochondria. Their proportion was unchanged in the N-acetylation mutant showing that neither modification affected the efficiency of import of adenylate kinase into mitochondria. It is discussed that N-acetylation occurs during or immediately after translation in the cytoplasm so that import of adenylate kinase may ensue co-translationally.

Acetylation↗

PubMed1996

Glucose-induced sequential processing of a glycosyl-phosphatidylinositol-anchored ectoprotein in Saccharomyces cerevisiae.

Transfer of spheroplasts from the yeast Saccharomyces cerevisiae to glucose leads to the activation of an endogenous (glycosyl)-phosphatidylinositol-specific phospholipase C ([G]PI-PLC), which cleaves the anchor of at least one glycosyl-phosphatidylinositol (GPI)-anchored protein, the cyclic AMP (cAMP)-binding ectoprotein Gce1p (G. Müller and W. Bandlow, J. Cell Biol. 122:325-336, 1993). Analyses of the turnover of two constituents of the anchor, myo-inositol and ethanolamine, relative to the protein label as well as separation of the two differently processed versions of Gce1p by isoelectric focusing in spheroplasts demonstrate the glucose-induced conversion of amphiphilic Gce1p first into a lipolytically cleaved hydrophilic intermediate, which is then processed into another hydrophilic version lacking both myo-inositol and ethanolamine. When incubated with unlabeled spheroplasts, the lipolytically cleaved intermediate prepared in vitro is converted into the version lacking all anchor constituents, whereby the anchor glycan is apparently removed as a whole. The secondary cleavage ensues independently of the carbon source, attributing the key role in glucose-induced anchor processing to the endogenous (G)PI-PLC. The secondary processing of the lipolytically cleaved intermediate of Gce1p at the plasma membrane is correlated with the emergence of a covalently linked high-molecular-weight form of a cAMP-binding protein at the cell wall. This protein lacks anchor components, and its protein moiety appears to be identical with double-processed Gce1p detectable at the plasma membrane in spheroplasts. The data suggest that glucose-induced double processing of GPI anchors represents part of a mechanism of regulated cell wall expression of proteins in yeast cells.

Carrier Proteins↗

PubMed1995

Strain-dependent occurrence of functional GTP:AMP phosphotransferase (AK3) in Saccharomyces cerevisiae.

The gene for yeast GTP:AMP phosphotransferase (PAK3) was found to encode a nonfunctional protein in 10 laboratory strains and one brewers' strain. The protein product showed high similarity to vertebrate AK3 and was located exclusively in the mitochondrial matrix. The deduced amino acid sequence revealed a protein that was shorter at the carboxyl terminus than all other known adenylate kinases. Introduction of a +1 frameshift into the 3'-terminal region of the gene extended homology of the deduced amino acid sequence to other members of the adenylate kinase family including vertebrate AK3. Frameshift mutations obtained after in vitro and in vivo mutagenesis were capable of complementing the adk1 temperature-conditional deficiency in Escherichia coli, indicating that the frameshift led to the expression of a protein that could phosphorylate AMP. Some yeasts, however, including strain D273-10B, two wine yeasts, and two more distantly related yeast genera, harbored an active allele, named AKY3, which contained a +1 frameshift close to the carboxyl terminus as compared with the laboratory strains. The encoded protein exhibited GTP:AMP and ITP:AMP phosphotransferase activities but did not accept ATP as phosphate donor. Although single copy in the haploid genome, disruption of the AKY3 allele displayed no phenotype, excluding the possibility that laboratory and brewers' strains had collected second site suppressors. It must be concluded that yeast mitochondria can completely dispense with GTP:AMP phosphotransferase activity.

Alleles↗

PubMed1995

Glucose induces amphiphilic to hydrophilic conversion of a subset of glycosyl-phosphatidylinositol-anchored ectoproteins in yeast.

Previously, we have studied the lipolytic cleavage of a glycosyl-phosphatidylinositol (GPI)-anchored plasma membrane protein in yeast in response to a physiologically relevant external signal, i.e., transfer of spehroplasts from lactate to glucose medium (cf. Müller and Bandlow (1993) J. Cell. Biol. 122, 325-336). In the present study the glucose-induced lipolytic processing of myo-[14C]inositol-labeled total GPI proteins of the plasma membrane and in particular of two such proteins, Gas1p and Gce1p, was examined in yeast spheroplasts. It was found that a small number of GPI proteins, among them Gce1p, are readily cleaved, whereas Gas1p and the majority of the GPI proteins are relatively little affected. Glucose-induced processing of Gce1pcould be demonstrated also in intact cells. Increased GPI cleavage after exposure of cells or spheroplasts to glucose is not due to stimulation of cell surface expression of Gce1p, as the amount of total GPI-anchored Gce1p bound to plasma membranes is comparable in cells grown in glucose or lactate. In agreement with this, Brefeldin A added together with the label blocks transport of newly made Gce1p to the cell surface and, in the consequence, cleavage of labeled Gce1p in spheroplasted yeast cells. (The drug is ineffective in intact cells). On the other hand, Brefeldin A does not significantly affect glucose-induced processing of inositol-labeled Gce1p at the plasma membrane when present during the period of nutritional upshift. We discuss that addition of glucose to the cells leads to the activation of a GPI-specific phospholipase which accepts only a subset of GPI proteins as substrates. This interpretation is additionally corroborated by the finding that purified [14C]inositol-labeled Gcep1p is lipolytically cleaved when incubated with Triton X-100-insoluble membrane complexes isolated from glucose-induced but not from uninduced spheroplasts. It is concluded that the phospholipase is present in these complexes and its state of activity is preserved during the preparation. GPI anchor cleavage in yeast appears to resemble strikingly the situation in insulin-responsive adipocytes subsequently to stimulation by insulin of glucose transport into these cells.

Biological Transport↗

PubMed1995

Selection of specific gene probes by combined use of low-stringency PCR amplification and Southern-blot hybridization.

We have developed a protocol to isolate a gene from which only limited (amino-acid) sequence information is available. It involves two PCR amplifications using one constant primer and a set of nested primers and subsequent crosswise Southern hybridization. The amplified DNA giving a signal in both lanes is further processed for use in gene bank screening by applying standard procedures. In this way the structural gene for a thiol protease, BLH1, the homologue of the bleomycin A (a cancerostatic drug) resistance gene of rabbit (and man), was isolated from yeast genomic DNA.

Amino Acid Sequence↗