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E Kübler

Publications and source records attributed to E Kübler.

12 recordsLinked to original sources
PubMed2001

Regulation of hisHF transcription of Aspergillus nidulans by adenine and amino acid limitation.

The hisHF gene of Aspergillus nidulans encodes imidazole-glycerole-phosphate (IGP) synthase, consisting of a glutamine amidotransferase and a cyclase domain. The enzyme catalyzes the fifth and sixth steps of histidine biosynthesis, which results in an intermediate of the amino acid and an additional intermediate of purine biosynthesis. An A. nidulans hisHF cDNA complemented a Saccharomyces cerevisiae his7Delta strain and Escherichia coli hisH and hisF mutant strains. The genomic DNA encoding the hisHF gene was cloned and its sequence revealed two introns within the 1659-bp-long open reading frame. The transcription of the hisHF gene of A. nidulans is activated upon amino acid starvation, suggesting that hisHF is a target gene of cross pathway control. Adenine but not histidine, both end products of the biosynthetic pathways connected by the IGP synthase, represses hisHF transcription. In contrast to other organisms HISHF overproduction did not result in any developmental phenotype of the fungus in hyphal growth or the asexual life cycle. hisHF overexpression caused a significantly reduced osmotic tolerance and the inability to undergo the sexual life cycle leading to acleistothecial colonies.

Adenine↗
PubMed2000

Developmental and metabolic regulation of the phosphoglucomutase-encoding gene, pgmB, of Aspergillus nidulans.

We have isolated the pgmB gene from Aspergillus nidulans, which encodes a phosphoglucomutase, one of the key enzymes in carbohydrate metabolism. The pgmB gene is located on chromosome VII and its ORF encodes 557 amino acids. Mutant phenotypes were analysed by expression of high levels of pgmB antisense RNA, which lead to a loss of detectable levels of sense RNA. Under conditions of antisense RNA expression, a 30% reduction in the growth rate was observed in comparison to wild-type. On the enzyme level, expression of pgmB antisense RNA resulted in a 35% reduction in total phosphoglucomutase activity. Two pgmB mRNAs were observed under all conditions tested and differ with respect to the location of the poly(A) site. Expression of pgmB driven by the GAL1 promoter in Saccharomyces cerevisiae complemented the growth phenotype of a pgm2delta mutant strain and suppressed the sensitivity of a gcn4delta mutant strain to amino acid starvation in the presence of galactose. Cultivation of A. nidulans in the presence of glucose or galactose as carbon source did not affect transcription of pgmB. However, amino acid starvation conditions resulted in a six-fold reduction in the level of pgmB mRNA, while genes for amino acid biosynthesis showed increased transcription. Transcription of pgmB was low during hyphal growth and in the sexual phase of development, but was significantly increased during the asexual stage of the A. nidulans life cycle.

Amino Acid Sequence↗
PubMed2000

The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system.

The tryptophan synthase-encoding gene, trpB, of Aspergillus nidulans was cloned and characterized. It was mapped to chromosome I, between the gene medA, which is required for sexual and asexual development, and an ORF encoding a protein with significant similarity to subunit B of vacuolar ATP synthases. The 5' untranslated region was found to be at least 142 nucleotides (nt) long, the poly(A) addition site was localized at position + 216 relative to the stop codon by sequencing of several independent cDNA clones. The trpB gene contains two exons separated by an intron of 105 nt, which is located close to the 5' end of the ORF. Directly upstream of the transcriptional start site, one well conserved potential binding site for the cross-pathway control transcriptional activator CPCA was found. The level of trpB transcript was shown to be regulated by cross-pathway control. A knockout mutant for trpB displays tryptophan auxotrophy, no trpB transcript is detectable, and development is perturbed to an extent that is dependent on the amount of tryptophan added to the medium. The trpB gene encodes a protein of 723 amino acids, with a calculated molecular weight of 77.6 kDa. The deduced amino acid sequence shows 72.6% similarity to the tryptophan synthase of Neurospora crassa. Most amino acid residues essential for catalytic activity in the tryptophan synthase of Salmonella typhimurium are conserved. The linker region joining the two domains of the enzyme is 13 residues longer than the longest connector found so far in tryptophan synthases from fungi.

Amino Acid Sequence↗
PubMed1999

Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae.

The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth.

Adenylyl Cyclases↗
PubMed1997

Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism.

The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells. In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein alpha-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action. In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase. Of the two yeast G-protein alpha-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation. In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein alpha-subunit leads to a defect in pseudohyphal development. Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p. Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP. In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain. Taken together, our results directly demonstrate that a G-protein alpha-subunit can regulate the growth and pseudohyphal development of S. cerevisiae via a cAMP-dependent mechanism. Heterologous expression of mammalian G-protein alpha-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting.

Cyclic AMP↗
PubMed1996

Identification of Triton X-100 insoluble membrane domains in the yeast Saccharomyces cerevisiae. Lipid requirements for targeting of heterotrimeric G-protein subunits.

Low density Triton X-100 insoluble (LDTI) membrane domains are found in most mammalian cell types. Previous biochemical and immunolocalization studies have revealed the presence of G-protein coupled receptors and heterotrimeric G-protein subunits (Galpha and Gbetagamma subunits) within these structures, implicating mammalian LDTI membrane domains in G-protein coupled signaling. Here, we present biochemical evidence that similar LDTI structures exist in a genetically tractable organism, the yeast Saccharomyces cerevisiae. Yeast LDTI membranes were purified based on the known biochemical properties of mammalian LDTI membranes: (i) their Triton X-100 insolubility; and (ii) their discrete buoyant density in sucrose gradients. As with purified mammalian LDTI membranes, these yeast LDTI membranes harbor the subunits of the heterotrimeric G-proteins (Galpha and Gbetagamma subunits). Other plasma membrane marker proteins (the plasma membrane H+-ATPase and a GPI-linked protein Gas1p) are preferentially excluded from these purified fractions. Mutational and genetic analyses were performed to define the requirements for the targeting of G-protein subunits to these yeast membrane domains. We find that the targeting of Galpha is independent of myristoylation, whereas targeting of Ggamma requires prenylation. Perhaps surprisingly, the targeting of Gbeta to this membrane domain did not require coexpression of Ggamma. It should now be possible to dissect the function of LDTI membrane domains using yeast as a model genetic system.

Cell Compartmentation↗
PubMed1995

Oligomeric structure of caveolin: implications for caveolae membrane organization.

A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization of these purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as "fishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.

Animals↗
PubMed1994

Calcium-independent calmodulin requirement for endocytosis in yeast.

We have recently shown that actin and fimbrin are required for the internalization step of endocytosis in yeast. Using a yeast strain with a temperature-sensitive allele of CMD1, encoding calmodulin, we demonstrate that this protein is also required for this process. Calmodulin mutants that have lost their high-affinity calcium binding sites are, however, able to carry out endocytosis normally. A mutation in Myo2p, an unconventional myosin that is a possible target of calmodulin, did not inhibit endocytosis. The function of calmodulin in endocytosis seems to be specific among membrane trafficking events, because the calmodulin mutants are not defective for biogenesis of soluble vacuolar hydrolases nor invertase secretion. Calmodulin does not seem to play a major role in the post-internalization steps of the endocytic pathway in yeast.

Actins↗
PubMed1993

Actin and fimbrin are required for the internalization step of endocytosis in yeast.

In Saccharomyces cerevisiae, alpha-factor is internalized by receptor-mediated endocytosis and transported via vesicular intermediates to the vacuole where the pheromone is degraded. Using beta-tubulin and actin mutant strains, we showed that actin plays a direct role in receptor-mediated internalization of alpha-factor, but is not necessary for transport from the endocytic intermediates to the vacuole. beta-tubulin mutant strains showed no defect in these processes. In addition, cells lacking the actin-binding protein, Sac6p, which is the yeast fimbrin homologue, are defective for internalization of alpha-factor suggesting that actin filament bundling might be required for this step. The actin dependence of endocytosis shows some interesting similarities to endocytosis from the apical membrane in polarized mammalian cells.

Actins↗
PubMedDate not supplied

Caveolae, transmembrane signalling and cellular transformation.

Caveolae are approximately 50-100 nm membrane micro-invaginations associated with the plasma membrane of a wide variety of cells. Although they were first identified in transmission electron micrographs approximately 40 years ago, their exact function(s) has remained controversial. Two well-established functions include: (1) the transcytosis of both large and small molecules across capillary endothelial cells and (2) the utilization of GPI-linked proteins to concentrate small molecules in caveolae for translocation to the cytoplasm (termed potocytosis). Recently, interest in a 'third' proposed caveolar function, namely transmembrane signalling, has been revived by the identification of caveolin--a transformation-dependent v-Src substrate and caveolar marker protein--and the isolation of caveolin-rich membrane domains from cultured cells. Here we will discuss existing evidence that suggests a role for caveolae in signalling events.

Biomarkers↗