Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell-cycle phase-dependent degradation in yeast.

Using co-immunoprecipitation combined with MS analysis, we identified the alpha' subunit of casein kinase 2 (CK2) as an interaction partner of the atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2 from Deltacka1 or Deltacka2 mutant extracts shows that Rio1p preferentially interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential and sufficient for this interaction.

SUT2 is a novel multicopy suppressor of low activity of the cAMP/protein kinase A pathway in yeast.

SUT2 was found in a screen for multicopy suppressors of the synthetic slow growth phenotype of a Deltaras2Deltagpa2 double deletion mutant. It failed, however, to cure the lethal phenotype of a Deltaras1Deltaras2 mutant suggesting that it acts upstream of Ras or in a parallel pathway. By testing cAMP-dependent reactions including the accumulation of storage carbohydrates, pseudohyphal differentiation, entry of meiosis as well as the measurement of FLO11 reporter activity we show that Sut2p modulates the activity of protein kinase A (PKA).

A nucleosome-free dG-dC-rich sequence element promotes constitutive transcription of the essential yeast RIO1 gene.

RIO1 is an essential gene that encodes a protein serine kinase and is transcribed constitutively at a very low level. Transcriptional activation of RIO1 dispenses with a canonical TATA box as well as with classical transactivators or specific DNA-binding factors. Instead, a dG-dC-rich sequence element, that is located 40 to 48 bp upstream the single site of mRNA initiation, is essential and presumably constitutes the basal promoter.

Structural determinants of the rate of protein folding.

To understand the mechanism of protein folding and to assist rational design of fast-folding, non-aggregating and stable artificial enzymes, it is essential to determine the structural parameters which govern the rate constants of folding, kf. It has been found that -logkf is a linear function of the so-called chain topology parameter (CTP) within the range of 10(-1)s(-1)< or = kf < or =10(8)s(-1). The correlation between -logkf and CTP is much improved than using previously published contact order (CO) method.

Reb1p-dependent DNA bending effects nucleosome positioning and constitutive transcription at the yeast profilin promoter.

The molecular basis of constitutive gene activation is largely unknown. The yeast profilin gene (PFY1), encoding a housekeeping component of the actin cytoskeleton, is constitutively transcribed at a moderate level. The PFY1 promoter dispenses with classical transactivators and a consensus TATA box; however, it contains a canonic site for the abundant multifunctional nuclear factor rDNA enhancer-binding protein (Reb1p) combined with a dA.

Permanent nucleosome exclusion from the Gal4p-inducible yeast GCY1 promoter.

The promoter of the galactose-inducible yeast GCY1 gene allows high rates of basal transcription and is kept free of nucleosomes regardless of growth conditions. The general regulatory factor, Reb1p, as well as the nucleotide sequence of a single Gal4p-binding site, structurally cooperate to exclude nucleosomes from about 480 bp of DNA that spans the UAS(GAL), the Reb1p-binding site, the TATA-box, and the transcriptional initiation sites. Gal4p, which induces transcription of GCY1 about 25-fold in the presence of galactose, is not required for the alteration in chromatin configuration in the promoter upstream region since the hypersensitive site is unchanged when Gal4p is inactive or absent.

Interaction of phosphoinositolglycan(-peptides) with plasma membrane lipid rafts of rat adipocytes.

Insulin receptor-independent activation of the insulin signal transduction cascade in insulin-responsive target cells by phosphoinositolglycans (PIG) and PIG-peptides (PIG-P) is accompanied by redistribution of glycosylphosphatidylinositol (GPI)-anchored plasma membrane proteins (GPI proteins) and dually acylated nonreceptor tyrosine kinases from detergent/carbonate-resistant glycolipid-enriched plasma membrane raft domains of high-cholesterol content (hcDIGs) to rafts of lower cholesterol content (lcDIGs). Here we studied the nature and localization of the primary target of PIG(-P) in isolated rat adipocytes. Radiolabeled PIG-P (Tyr-Cys-Asn-NH-(CH(2))(2)-O-PO(OH)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manalpha1-4GluN1-6Ino-1,2-(cyclic)-phosphate) prepared by chemical synthesis or a radiolabeled lipolytically cleaved GPI protein from Saccharomyces cerevisiae, which harbors the PIG-P moiety, bind to isolated hcDIGs but not to lcDIGs.

Interaction of phosphatidylinositolglycan(-peptides) with plasma membrane lipid rafts triggers insulin-mimetic signaling in rat adipocytes.

The phosphoinositolglycan(-peptide) (PIG-P) portion of glycosylphosphatidylinositol-anchored plasma membrane (GPI) proteins or synthetic PIG(-P) molecules interact with proteinaceous binding sites which are located in high-cholesterol-containing detergent/carbonate-insoluble glycolipid-enriched raft domains (hcDIGs) of the plasma membrane. In isolated rat adipocytes, PIG(-P) induce the redistribution of GPI proteins from hcDIGs to low-cholesterol-containing DIGs (lcDIGs) and concomitantly provoke insulin-mimetic signaling and metabolic action. Using a set of synthetic PIG(-P) derivatives we demonstrate here that their specific binding to hcDIGs and their insulin-mimetic signaling/metabolic activity strictly correlate with respect to (i) translocation of the GPI proteins, Gce1 and 5(')-nucleotidase, from hcDIGs to lcDIGs, (ii) dissociation of the nonreceptor tyrosine kinase, pp59(Lyn), from caveolin residing at hcDIGs, (iii) translocation of pp59(Lyn) from hcDIGs to lcDIGs, (iv) activation of pp59(Lyn), (v) tyrosine phosphorylation of insulin receptor substrate proteins-1/2, and finally (vi) stimulation of glucose transport.

Transcription initiation in vivo without classical transactivators: DNA kinks flanking the core promoter of the housekeeping yeast adenylate kinase gene, AKY2, position nucleosomes and constitutively activate transcription.

The housekeeping gene of the major adenylate kinase in Saccharomyces cerevisiae (AKY2, ADK1) is constitutively transcribed at a moderate level. The promoter has been dissected in order to define elements that effect constitutive transcription. Initiation of mRNA synthesis at the AKY2 promoter is shown to be mediated by a non-canonic core promoter, (TA)(6).

RIO1, an extraordinary novel protein kinase.

RIO1 and Rio-related proteins display little similarity of primary sequence with conventional protein kinases. Based on secondary structure alignments, we show that it contains the domain structure (subdomains I-XI) and conserved secondary structure elements found in conventional protein kinases. We show that recombinant wild-type Rio1p isolated from Escherichia coli displays kinase activity which depends on autophosphorylation and magnesium or manganese as ATP-activating ions.

Redundant mitochondrial targeting signals in yeast adenylate kinase.

Yeast adenylate kinase (Aky2p, Adk1p) occurs simultaneously in cytoplasm and mitochondrial intermembrane space. It has no cleavable mitochondrial targeting sequence, and the signal for mitochondrial import and submitochondrial sorting is largely unknown. The extreme N terminus of Aky2p is able to direct cytoplasmic passengers to mitochondria.

Competition of spontaneous protein folding and mitochondrial import causes dual subcellular location of major adenylate kinase.

Sorting of cytoplasmically synthesized proteins to their target compartments usually is highly efficient so that cytoplasmic precursor pools are negligible and a particular gene product occurs at one subcellular location only. Yeast major adenylate kinase (Adk1p/Aky2p) is one prominent exception to this rule. In contrast to most mitochondrial proteins, only a minor fraction (6-8%) is taken up into the mitochondrial intermembrane space, whereas the bulk of the protein remains in the cytosol in sequence-identical form.

Yeast Rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression.

Rio1p was identified as a protein serine kinase founding a novel subfamily. It is highly conserved from Archaea to man and only distantly related to previously established protein kinase families. Nevertheless, analysis of multiple protein sequence alignments shows that those amino acid residues that are important for either structure or catalytic activity in conventional protein kinases are also conserved in members of the Rio1p family at the respective positions (corresponding to domains I-XI of protein kinases).