Biomedical subjects

W Bandlow

Publications and source records attributed to W Bandlow.

At least 37 records · Page 2Linked to original sources

PubMed1994

Membrane association of lipoprotein lipase and a cAMP-binding ectoprotein in rat adipocytes.

cAMP-binding ectoprotein (Gce1) and lipoprotein lipase (LPL) are anchored to plasma membranes of rat adipocytes by glycosylphosphatidylinositol (GPI) moieties as demonstrated by cleavage by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), reactivity with anti-crossreacting determinant antibodies (anti-CRD), and metabolic labeling with radiolabeled palmitic acid and myo-inositol. Quantitative release from the membrane of LPL and Gce1 requires both lipolytic removal of their GPI anchors and the presence of either 2 M NaCl or 1 mM inositol 1,2-cyclic monophosphate or inositol 1-monophosphate. PI-PLC-cleaved and released LPL or Gce1 reassociates with isolated plasma membranes of rat adipocytes and, less efficiently, with membranes of 3T3 fibroblasts. The specificity of the reassociation is demonstrated (i) by its inhibition after pretreatment of the membranes with trypsin, (ii) by its competition with inositol 1,2-cyclic monophosphate and inositol 1-monophosphate in a concentration-dependent manner, and (iii) by the limited number of binding sites. Enzymic or chemical removal as well as masking with anti-CRD antibodies of the terminal inositol (cyclic) monophosphate moiety of hydrophilic Gce1 and LPL significantly impairs the reassociation. These data suggest that in rat adipocytes GPI-proteins are not readily released from the cell surface upon lipolytic cleavage, but remain associated through a receptor which specifically recognizes the terminal inositol (cyclic) monophosphate epitope of the (G)PI-PLC-cleaved GPI moiety. This interaction may have implications for the regulated membrane release of GPI-proteins and for their possible internalization.

3T3 Cells↗

PubMed1994

SWH1 from yeast encodes a candidate nuclear factor containing ankyrin repeats and showing homology to mammalian oxysterol-binding protein.

The isolation of a gene from Saccharomyces cerevisiae, SWH1, with a coding capacity for a 135 kDa protein is reported. The deduced amino acid sequence is homologous to mammalian oxysterol-binding protein (33.6% identical residues at homologous positions) but, in addition, predicts several structural modules that are not present in the mammalian counterpart. These comprise two ankyrin repeats as an N-terminal extension, and highly acidic clusters, poly-asparagine tracts as well as domains that constitute presumptive nuclear targeting signals interspersed in the N-terminal half of the yeast protein. The gene is transcribed constitutively at a low level from a promoter lacking an obvious TATA element. Heterozygous chromosomal deletion of the gene in a diploid yeast strain has no effect on sporulation or on germination of the four spores from one tetrad nor do haploid deletion mutants display any obvious disadvantage regarding growth behaviour or mating.

Amino Acid Sequence↗

PubMed1994

The gene LEO1 on yeast chromosome XV encodes a non-essential, extremely hydrophilic protein.

A 5.6 kbp segment of DNA from Saccharomyces cerevisiae chromosome XV has been isolated and sequenced. Genetic and nucleotide sequence analyses revealed that this region is closely linked to the ADE2 marker on chromosome XV and densely packed with genetic information. We show the gene organization of the entire region and report the nucleotide sequence of the gene, LEO1, which occurs in single copy in the haploid genome. The deduced amino acid sequence specifies an extremely hydrophilic protein with pronounced domain structure (molecular mass 53.9 kDa). The gene is constitutively expressed at a low level and is non-essential, as indicated by the absence of a phenotype from gene disruption mutants.

Amino Acid Sequence↗

PubMed1994

Lipolytic membrane release of two phosphatidylinositol-anchored cAMP receptor proteins in yeast alters their ligand-binding parameters.

Two new cAMP-binding proteins have been discovered recently in Saccharomyces cerevisiae. They are genetically distinct from the regulatory subunit of cytoplasmic cAMP-dependent protein kinase A and are distinguished from the latter, in addition, by their anchorage through phosphatidylinositol-containing lipid and glycolipid structures to mitochondrial and plasma membranes, respectively (Müller and Bandlow, 1989 Biochemistry 28, 9957-9967, 1991, Biochemistry 30, 10181-10190). A nutritional upshift induces the cleavage of the anchor by a phospholipase C (Müller and Bandlow, 1993, J. Cell Biol. 122, 225-236). To test the idea that anchorage by (glycosyl)phosphatidyl-inositol influences cAMP-binding and has a regulatory function, we analyzed ligand binding to the two purified cAMP receptors (46,000 and 54,000 Da) in comparison to the regulatory subunit of the cytoplasmic protein kinase A (52,000 Da). We find that lipolytic cleavage of the two membrane anchors by phosphatidylinositol-specific phospholipases C and D results in significantly higher association and lower dissociation rates of cAMP, thus leading to a dramatic increase in ligand affinity of the two cAMP receptors. Use of cAMP analogues identifies two different cAMP-binding centers in each membrane-embedded protein, one of which is noticeably affected by the cleavage of the anchor. In both phosphatidylinositol-anchored cAMP receptor proteins a single Trp residue in one of the binding centers is photoaffinity-labeled by 8-N3-cAMP, whereas two amino acids, Trp and Tyr, are modified after lipolytic removal of the anchor. The differences in the labeling patterns are interpreted as to result from a conformational rearrangement induced by the cleavage of the anchor. Together with the increased affinity to the ligand these changes document alterations of the properties and folding structure of lipid-anchored proteins following cleavage of the PI-containing anchor by specific phospholipases and provide the first molecular evidence for a regulatory role of the anchorage by a lipid structure. The cytoplasmic regulatory subunit of yeast protein kinase A is not photolabeled to a significant extent under any condition.

Affinity Labels↗

PubMed1994

Stimulation of a glycosyl-phosphatidylinositol-specific phospholipase by insulin and the sulfonylurea, glimepiride, in rat adipocytes depends on increased glucose transport.

Lipoprotein lipase (LPL) and glycolipid-anchored cAMP-binding ectoprotein (Gce1) are modified by glycosyl-phosphatidylinositol (GPI) in rat adipocytes, however, the linkage is potentially unstable. Incubation of the cells with either insulin (0.1-30 nM) or the sulfonylurea, glimepiride (0.5-20 microM), in the presence of glucose led to conversion of up to 35 and 20%, respectively, of the total amphiphilic LPL and Gce1 to their hydrophilic versions. Inositol-phosphate was retained in the residual protein-linked anchor structure. This suggests cleavage of the GPI anchors by an endogenous GPI-specific insulin- and glimepiride-inducible phospholipase (GPI-PL). Despite cleavage, hydrophilic LPL and Gce1 remained membrane associated and were released only if a competitor, e.g., inositol-(cyclic)monophosphate, had been added. Other constituents of the GPI anchor (glucosamine and mannose) were less efficient. This suggests peripheral interaction of lipolytically cleaved LPL and Gce1 with the adipocyte cell surface involving the terminal inositol-(cyclic)monophosphate epitope and presumably a receptor of the adipocyte plasma membrane. In rat adipocytes which were resistant toward glucose transport stimulation by insulin, the sensitivity and responsiveness of GPI-PL to stimulation by insulin was drastically reduced. In contrast, activation of both GPI-PL and glucose transport by the sulfonylurea, glimepiride, was not affected significantly. Inhibition of glucose transport or incubation of rat adipocytes in glucose-free medium completely abolished stimulation of GPI-PL by either insulin or glimepiride. The activation was partially restored by the addition of glucose or nonmetabolizable 2-deoxyglucose. These data suggest that increased glucose transport stimulates a GPI-PL in rat adipocytes.

Adipocytes↗

PubMed1993

The ethanol-inducible YAT1 gene from yeast encodes a presumptive mitochondrial outer carnitine acetyltransferase.

The gene YAT1 from Saccharomyces cerevisiae encodes a protein of 688 amino acids which displays significant sequence similarity to vertebrate L-carnitine acyltransferases and yeast inner mitochondrial L-carnitine acetyltransferase. Steady state levels of the respective mRNA are low during growth on glucose or galactose, derepressed on glycerol, and significantly induced when ethanol or acetate are the only carbon sources. The YAT1 promotor region confers the identical carbon source dependence also on the expression of beta-galactosidase when cloned 5' to the Escherichia coli lacZ-coding region. An antiserum directed against a beta-galactosidase/Yat1p fusion protein recognizes a protein of 78-kDa molecular mass in the mitochondrial fraction from yeast. In vitro translated Yat1p, which lacks a cleavable presequence, binds to mitochondria in a protease-sensitive location in a standard in vitro import assay. Deletion of the single copy gene in a haploid yeast strain yields no obvious phenotype with any carbon source. Carnitine acetyltransferase activities of intact mitochondria from a YAT1 deletion mutant are slightly lower than wild type, but are approximately alike in lysed mitochondria from mutant and control cells. Thus, the novel gene is nonessential and likely to code for a minor mitochondrial outer carnitine acetyltransferase.

Amino Acid Sequence↗

PubMed1993

A yeast gene (BLH1) encodes a polypeptide with high homology to vertebrate bleomycin hydrolase, a family member of thiol proteinases.

We have purified bleomycin hydrolase from yeast (molecular mass 55,000 Da). Using protein sequence-derived degenerate oligonucleotide primers and amplification by polymerase chain reaction, the yeast gene BLH1 was isolated and characterized. The deduced amino acid sequence (483 amino acids) exhibits surprisingly high homology to vertebrate bleomycin hydrolase (43% identical residues and 22% conserved exchanges). It contains three blocks of sequences found conserved in other members of the thiol proteinase family and thought to be associated with the catalytic centre. BLH1 is non-essential under all growth conditions tested. However, in the presence of 3.5 mg bleomycin/ml medium wild-type cells have a slight growth advantage compared to blh1 mutant cells.

Amino Acid Sequence↗

PubMed1993

High levels of profilin suppress the lethality caused by overproduction of actin in yeast cells.

Overproduction of actin is lethal to yeast cells. In contrast, overexpression of the profilin gene, PFY1, encoding an actin-binding protein, leads to no very obvious phenotype. Interestingly, profilin overproduction can compensate for the deleterious effects of too much actin in a profilin concentration-dependent manner. Our results, thus, document that actin and profilin interact in vivo. Immunofluorescence studies suggest that suppression works by reducing actin assembly. We observed, however, that even massive overproduction of profilin fails to fully restore the wild-type phenotype (e.g. the wild-type appearance of the actin microfilament system). This may indicate that actin monomer sequestration is not the only mechanism by which the balance of actin polymerization is controlled.

Actins↗

PubMed1993

Glucose induces lipolytic cleavage of a glycolipidic plasma membrane anchor in yeast.

In the yeast Saccharomyces cerevisiae an amphiphilic cAMP-binding protein has been found recently to be anchored to plasma membranes by virtue of a glycolipid structure (Müller and Bandlow, 1991a, 1992). The cAMP-binding parameters of this protein are affected by the lipolytic removal of the glycosylphosphatidylinositol (GPI) membrane anchor by exogenous (G)PI-specific phospholipases C or D (PLC or PLD) (Müller and Bandlow, 1993) suggesting a regulatory role of glycolipidic membrane anchorage. Here we report that transfer of yeast cells from lactate to glucose medium results in the conversion of the amphiphilic form of the cAMP receptor protein into a hydrophilic version accompanied by the rapid loss of fatty acids from the GPI anchor of the [14C]palmitic acid-labeled protein. Analysis of the cleavage site identifies [14C]inositol phosphate as the major product after treatment of the soluble, [14C]inositol-labeled protein with nitrous acid which destroys the glucosamine constituent of the anchor. Together with the observed cross-reactivity of the hydrophilic fragment with antibodies directed against the cross-reacting determinant of soluble trypanosomal variable surface glycoproteins (i.e., myo-inositol-1,2-cyclic phosphate) this demonstrates that, in membrane release, the initial cleavage event is catalyzed by an intrinsic GPI-PLC activated upon transfer of cells to glucose medium. Release from the plasma membrane in soluble form requires, in addition, the presence of high salt or alpha-methyl mannopyranoside, or the removal of the carbohydrate moieties, because otherwise the protein remains associated with the membrane presumably at least in part via its N-glycosidic carbohydrate side chains. The data point to the possibility that cleavage of the anchor could play a role in the transfer of the signal for the nutritional situation to the interior of the cell.

Carrier Proteins↗

PubMed1992

The cAMP-binding ectoprotein from Saccharomyces cerevisiae is membrane-anchored by glycosyl-phosphatidylinositol.

Saccharomyces cerevisiae contains an amphiphilic cAMP-binding glycoprotein at the outer face of the plasma membrane (M(r) = 54,000). It is converted to a hydrophilic form by treatment with glycosyl-phosphatidylinositol-specific phospholipases C and D (GPI-PLC/D), suggesting membrane anchorage by a covalently bound glycolipid. Determination of the constituents of the purified anchor by gas-liquid chromatography and amino acid analysis reveals the presence of glycerol, myo-inositol, glucosamine, galactose, mannose, ethanolamine, and asparagine (as the carboxyl-terminal amino acid of the Pronase-digested protein to which the anchor is attached). Complementary results are obtained by metabolic labeling, indicating that fatty acids and phosphorus are additional anchor constituents. The phosphorus is resistant to alkaline phosphatase, whereas approximately half is lost from the protein after treatment with GPI-PLD or nitrous acid, and all is removed by aqueous HF indicating the presence of two phosphodiester bonds. Inhibition of N-glycosylation by tunicamycin or removal of protein-bound glycan chains by N-glycanase or Pronase does not abolish radiolabeling of the anchor structure by any of the above compounds. Analysis of the products obtained after sequential enzymic and chemical degradation of the anchor agrees with the arrangement of constituents in GPIs from higher eucaryotes. Evidence for anchorage of the yeast cAMP-binding protein by a GPI anchor is strengthened additionally by the reactivity of the GPI-PLC-cleaved anchor with antibodies directed against the cross-reacting determinant of trypanosomal variant surface glycoproteins.

Amidohydrolases↗

PubMed1992

The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase.

The gene URA6 encoding uridylate kinase (UK) from Saccharomyces cerevisiae was isolated as a multicopy suppressor of the respiratory-deficient phenotype of an S. cerevisiae mutant defective in the gene AKY2 encoding AMP kinase (AK). The URA6 gene also restored temperature resistance to two different temperature-sensitive mutations in the gene encoding Escherichia coli AK. By contrast, the gene encoding UK of Dictyostelium discoideum on a multicopy yeast shuttle plasmid, expressed under control of the constitutive yeast AKY2 promoter, failed to complement the deficiency in yeast, although such transformants expressed high UK activity. We show that yeast UK exerts significant AK activity which is responsible for the complementation and is absent in the analogous enzyme from D. discoideum. Since UK also significantly phosphorylates CMP (but not GMP), it must be considered an unspecific short-form nucleoside monophosphate kinase. Wild-type mitochondria lack UK activity, but import AKY2. Since multicopy transformation with URA6 heals the Pet- phenotype of AKY2 disruption mutants, the presence of AKY2 in the mitochondrial intermembrane space is not required to maintain respiratory competence. However, furnishing UK with the bipartite intermembrane space-targeting presequence of cytochrome c1 improves the growth rates of AKY2 mutants with nonfermentable substrates, suggesting that AK activity in mitochondria is helpful, though not essential for oxidative growth.

Adenylate Kinase↗

PubMed1992

pYLZ vectors: Saccharomyces cerevisiae/Escherichia coli shuttle plasmids to analyze yeast promoters.

Two yeast/Escherichia coli shuttle vectors have been constructed to analyze promoter structures in Saccharomyces cerevisiae: the multicopy vector, pYLZ-2, and the centromere-based vector, pYLZ-6. Both plasmids contain the coding region of lacZ from E. coli lacking the N-terminal eight amino acids. The truncated reporter gene is preceded by a short polylinker (MCS) suitable for the insertion of promoter fragments. The vectors allow for the study of expression from complete promoters containing UAS and TATA elements, transcriptional start point(s) and the original context of the ATG start codon of a yeast gene. A yeast terminator fragment has been inserted 3' of the lacZ coding region. It contains the transcription termination region of the convergently transcribed yeast genes, GCY1 and PFY1, together with sequences corresponding to the mapped 3'-ends of the respective mRNAs. As an example, reporter activity was measured with promoter fragments from three yeast genes (GCY1, PFY1 and LEO1). The results demonstrate the efficiency of the plasmids for studying constitutive and regulated transcription, both at high and low levels of expression.

Base Sequence↗

PubMed1992

In vivo import of yeast adenylate kinase into mitochondria affected by site-directed mutagenesis.

Site-directed mutagenesis and deletions were used to study mitochondrial import of a major yeast adenylate kinase, Aky2p. This enzyme lacks a cleavable presequence and occurs in active and apparently unprocessed form both in mitochondria and cytoplasm. Mutations were applied to regions known to be surface-exposed and to diverge between short and long isoforms. In vertebrates, short adenylate kinase isozymes occur exclusively in the cytoplasm, whereas long versions of the enzyme have mitochondrial locations. Mutations in the extra loop of the yeast (long-form) enzyme did not affect mitochondrial import of the protein, whereas variants altered in the central, N- or C-terminal parts frequently displayed increased or, in the case of a deletion of the 8 N-terminal triplets, decreased import efficiencies. Although the N-terminus is important for targeting adenylate kinase to mitochondria, other parameters like internal sequence determinants and folding velocity of the nascent protein may also play a role.

Adenylate Kinase↗

PubMed1992

A new member of the adenylate kinase family in yeast: PAK3 is highly homologous to mammalian AK3 and is targeted to mitochondria.

Making use of the polymerase chain reaction primed by oligonucleotides corresponding to regions conserved between members of the nucleoside monophosphate kinase family, we have isolated the yeast gene PAK3. Pak3p belongs to the subgroup of long-form adenylate kinase isozymes (deduced molecular mass 25.3 kDa) and exhibits highest sequence similarity to bovine AK3 rather than to the yeast isozyme, Aky2p. The gene is shown to be non-essential because haploid disruption mutants are viable, both in the presence and absence of a functional AKY2 allele. It maps on chromosome V upstream of RAD3. Its expression level is low when cells are grown on glucose or other fermentable carbon sources and about threefold higher on glycerol, but can be significantly induced by ethanol. A PAK3/mouse dihydrofolate reductase fusion construct expressed in yeast is targeted to mitochondria. Transformation with PAK3 on a multicopy plasmid complements neither adenylate kinase deficiency in an aky2-disrupted yeast strain nor in Escherichia coli cells conditionally defective in adenylate kinase.

Adenylate Kinase↗

PubMed1992

Expression of yeast cytochrome c1 is controlled at the transcriptional level by glucose, oxygen and haem.

The nuclear gene for cytochrome c1 in Saccharomyces cerevisiae (CYT1) was localized on chromosome XV. Its upstream region was identified by functional complementation. Fusion to the lacZ reporter gene on a CEN plasmid allowed study of the effect of carbon sources and of specific deletion mutations on expression of the gene in yeast transformants. Detailed promoter analysis combined with expression studies in recipient strains defective in regulatory genes identified cis-acting sites and transcription factors involved in the regulated expression of the cytochrome c1 gene. These analyses showed that, in the presence of glucose, transcription of CYT1 is positively controlled by oxygen, presumably through the haem signal, and mediated by the HAP1-encoded transactivator. It is additionally regulated by the HAP2/3/4 complex which mediates gene activation mainly under glucose-free conditions. Basal transcription is, in part, effected by CPF1, a centromere and promoter-binding factor.

Base Sequence↗

PubMed1991

Two lipid-anchored cAMP-binding proteins in the yeast Saccharomyces cerevisiae are unrelated to the R subunit of cytoplasmic protein kinase A.

We show that the yeast, Saccharomyces cerevisiae, contains two cAMP-binding proteins in addition to the well-characterized regulatory (R) subunit of cytoplasmic cAMP-dependent protein kinase (PKA). We provide evidence that they comprise a new type of cAMP receptor, membrane-anchored by covalently attached lipid structures. They are genetically not related to the cytoplasmic R subunit. The respective proteins can be detected in sral mutants, in which the gene for the R subunit of PKA has been disrupted and a monoclonal antibody raised against the cytoplasmic R subunit does not cross-react with the two membrane-bound cAMP-binding proteins. In addition, they differ from the cytoplasmic species also with respect to their location and the peptide maps of the photoaffinity-labeled proteins. Although they differ from one another in molecular mass and subcellular location, peptide maps of the cAMP-binding domains resemble each other and both proteins are membrane-anchored by lipid structures, one to the outer surface of the plasma membrane, the other to the outer surface of the inner mitochondrial membrane. Both anchors can be metabolically labeled by Etn, myo-Ins and fatty acids. In addition, the anchor structure of the cAMP receptor from plasma membranes can be radiolabeled by GlcN and Man. After cleavage of the anchor with glycosylphosphatidylinositol-specific phospholipase C from trypanosomes, the solubilized cAMP-binding protein from plasma membranes reacts with antibodies which specifically recognize the cross-reacting determinant from soluble trypanosomal coat protein, suggesting similarity of the anchors. Degradation studies also point to the glycosylphosphatidylinositol nature of the anchor from the plasma membrane, whereas the mitochondrial counterpart is less complex in that it lacks carbohydrates. The plasma membrane cAMP receptor is, in addition, modified by an N-glycosidically linked carbohydrate side chain, responsible mainly for its higher molecular mass.

Affinity Labels↗

PubMed1991

A cAMP-binding ectoprotein in the yeast Saccharomyces cerevisiae.

Purified plasma membranes from the yeast Saccharomyces cerevisiae bind about 1.2 pmol of cAMP/mg of protein with high affinity (Kd = 6 nM). By using photoaffinity labeling with 8-N3-[32P]cAMP, we have identified in plasma membrane vesicles a cAMP-binding protein (Mr = 54,000) that is present also in bcy1 disruption mutants, lacking the cytoplasmic R subunit of protein kinase A (PKA). This argues that it is genetically unrelated to PKA. Neither high salt, nor alkaline carbonate, nor cAMP extract the protein from the membrane, suggesting that it is not peripherally bound. The observation that (glycosyl)phosphatidylinositol-specific phospholipases (or nitrous acid) release the amphiphilic protein from the membrane, thereby converting it to a hydrophilic form, indicates anchorage by a glycolipidic membrane anchor. Treatment with N-glycanase reduces the Mr to 44,000-46,000 indicative of a modification by N-linked carbohydrate side chain(s). In addition to the action of a phospholipase, the efficient release from the membrane requires the removal of the carbohydrate side chain(s) or the presence of high salt or methyl alpha-mannopyranoside, suggesting complex interactions with the membrane involving not only the glycolipidic anchor but also the glycan side chain(s). Topological studies show that the protein is exposed to the periplasmic space, raising intriguing questions for the function of this protein.

Carbohydrates↗

PubMed1990

Transcriptional control by galactose of a yeast gene encoding a protein homologous to mammalian aldo/keto reductases.

Expression of the S. cerevisiae gene, GCY, encoding a 35-kDa protein with striking homology to mammalian aldo/keto reductases, is under the control of galactose: the intracellular concentration of the respective mRNA (about 1300 nt in length) varies strongly with the carbon source. It is particularly high when galactose is the sole energy source but is low as soon as glucose is present. Lactate, glycerol and raffinose lead to intermediate expression. Both Northern blot analyses and lacZ fusion data indicate a 20- to 50-fold increase in the steady state concentrations of mRNA and beta Gal activity, respectively, when grown on galactose as compared to glucose. The gene is derepressed after cultivation on glycerol in the wt and in a gal80 mutant background but remains uninducible by galactose in strains carrying either a gal2 or a gal4 mutation, affecting galactose permease and the GAL gene trans-activator, respectively. Analysis of GCY expression in gal regulatory mutants reveals epistasis interactions of the gal4 and the gal80 mutations as expected if GCY is regulated by the Gal control system. Repression of GCY transcription by glucose is observed in all three above gal mutant strains. The results suggest that the gene is both positively controlled by galactose and negatively by glucose. Analysis of a set of upstream deletions identifies a single UAS matching the consensus for GAL gene upstream regulation sites. By contrast to other genes regulated by galactose, disruption mutants of GCY exhibit no obvious phenotype, and in particular do not lose the ability to grow on and adapt to galactose. Enzyme tests with AKR-specific substrates suggest that GCY encodes a carbonyl reductase.

Base Sequence↗