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S Keränen

Publications and source records attributed to S Keränen.

At least 19 recordsLinked to original sources
PubMed2001

The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae.

The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19-1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19-1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes.

Amino Acid Sequence↗
PubMed2001

Characterization of the sec1-1 and sec1-11 mutations.

Sec1 proteins are implicated in positive and negative regulation of SNARE complex formation. To better understand the function of Sec1 proteins we have identified the nature of the temperature-sensitive mutations in sec1-1 and sec1-11. The sec1-1 mutation changes a conserved glycine(443) to glutamic acid. The sec1-11 mutation changes a highly conserved arginine(432) to proline. Based on homology and the crystal structure of the mammalian nSec1p, the corresponding amino acids localize to the 3b domain of nSec1p. Compared to the wild-type Sec1p the mutant proteins are less abundant even at the permissive temperature. Thus, the R432P and G443E mutations may cause structural alterations that affect folding and make the mutant proteins more susceptible to degradation. The remaining part is sufficient for growth and protein secretion at 24 degrees C and thus is likely to be properly folded. At 37 degrees C the mutant proteins become non-functional. In pulse-chase-type experiments the newly synthesized Sec1-1 and Sec1-11 proteins decayed similarly with the wild-type protein. Thus, the non-functionality of the mutant proteins cannot be explained by denaturation-induced degradation only. It is possible that the newly synthesized mutant proteins fold slowly and are susceptible to degradation before they have managed to fold and associate with other proteins. The mutant proteins were unable to interact with the Sec1p-interacting proteins Mso1p and Sso2p in the two-hybrid assay, even at the permissive temperature. These results localize sec1-1 and sec1-11 mutations to a domain of Sec1p and suggest a mechanism by which sec1-1 and sec1-11 cells become temperature-sensitive.

Amino Acid Sequence↗
PubMed2001

Trichoderma reesei rho3 a homologue of yeast RH03 suppresses the growth defect of yeast sec15-1 mutation.

The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast. The encoded protein showed 61% amino acid identity to the Rho3 protein. Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S. cerevisiae RHO3 gene. In addition, overproduction of T. reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells. Three putative mutant (rho3Gly20Val alleles of the T. reesei rho3 gene rho3 Thr25Asn, rho3Asp12Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion. Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3 Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype.

Amino Acid Sequence↗
PubMed2001

Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei.

We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature-sensitive secretory mutation, sec15-1. To characterize this gene further, we tested its ability to suppress other late-acting secretory mutations. The growth defect of yeast strains with sec1-1, sec1-11, sec3-2, sec6-4 and sec8-9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15-1 cells at +38 degrees C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3-101, sec5-24, sec8-9, sec10-2 and sec15-1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3-101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei.

Base Sequence↗
PubMed2001

Enamel knots as signaling centers linking tooth morphogenesis and odontoblast differentiation.

Odontoblasts differentiate from the cells of the dental papilla, and it has been well-established that their differentiation in developing teeth is induced by the dental epithelium. In experimental studies, no other mesenchymal cells have been shown to have the capacity to differentiate into odontoblasts, indicating that the dental papilla cells have been committed to odontoblast cell lineage during earlier developmental stages. We propose that the advancing differentiation within the odontoblast cell lineage is regulated by sequential epithelial signals. The first epithelial signals from the early oral ectoderm induce the odontogenic potential in the cranial neural crest cells. The next step in the determination of the odontogenic cell lineage is the development of the dental papilla from odontogenic mesenchyme. The formation of the dental papilla starts at the onset of the transition from the bud to the cap stage of tooth morphogenesis, and this is regulated by epithelial signals from the primary enamel knot. The primary enamel knot is a signaling center which forms at the tip of the epithelial tooth bud. It becomes fully developed and morphologically discernible in the cap-stage dental epithelium and expresses at least ten different signaling molecules belonging to the BMP, FGF, Hh, and Wnt families. In molar teeth, secondary enamel knots appear in the enamel epithelium at the sites of the future cusps. They also express several signaling molecules, and their formation precedes the folding and growth of the epithelium. The differentiation of odontoblasts always starts from the tips of the cusps, and therefore, it is conceivable that some of the signals expressed in the enamel knots may act as inducers of odontoblast differentiation. The functions of the different signals in enamel knots are not precisely known. We have shown that FGFs stimulate the proliferation of mesenchymal as well as epithelial cells, and they may also regulate the growth of the cusps. We have proposed that the enamel knot signals also have important roles, together with mesenchymal signals, in regulating the patterning of the cusps and hence the shape of the tooth crown. We suggest that the enamel knots are central regulators of tooth development, since they link cell differentiation to morphogenesis.

Animals↗
PubMed2000

Exocytosis requires asymmetry in the central layer of the SNARE complex.

Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

Alleles↗
PubMed2000

Role of adenine nucleotide translocator 1 in mtDNA maintenance.

Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.

Amino Acid Sequence↗
PubMed2000

Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast.

Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.

Biological Transport↗
PubMed1999

SEM1, a homologue of the split hand/split foot malformation candidate gene Dss1, regulates exocytosis and pseudohyphal differentiation in yeast.

The exocyst is an essential multiprotein complex mediating polarized secretion in yeast. Here we describe a gene, SEM1, that can multicopy-suppress exocyst mutants sec3-2, sec8-9, sec10-2, and sec15-1. SEM1 is highly conserved among eukaryotic species. Its human homologue, DSS1, has been suggested as a candidate gene for the split hand/split foot malformation disorder. SEM1 is not an essential gene. However, its deletion rescued growth of the temperature-sensitive exocyst mutants sec3-2, sec8-9, sec10-1, and sec15-1 at the restrictive temperature. Cell fractionation showed that Sem1p is mainly cytosolic but also associates with the microsomal fraction. In linear sucrose gradients, Sem1p cosedimented with the exocyst component Sec8p. In diploid cells that normally do not form pseudohyphae (S288C background), deletion of SEM1 triggered pseudohyphal growth. This phenotype was abolished after reintroduction of either SEM1 or the mouse homologue Dss1 into the cells. In diploids that have normal capacity for pseudohyphal growth (Sigma1278b background), deletion of SEM1 enhanced filamentous growth. The functionality of both SEM1 and Dss1 in a differentiation process in yeast suggests that Dss1 indeed could be the gene affected in the split hand/split foot malformation disorder. These results characterize SEM1 as a regulator of both exocyst function and pseudohyphal differentiation and suggest a unique link between these two cellular functions in yeast.

Amino Acid Sequence↗
PubMed1999

DNA polymerase epsilon catalytic domains are dispensable for DNA replication, DNA repair, and cell viability.

DNA polymerase epsilon (Pol epsilon) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol epsilon is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion of budding yeast Pol epsilon (Pol2) containing all known DNA polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability. However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability. Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.

Alleles↗
PubMed1998

Modes of action on cotton and bacterial cellulose of a homologous endoglucanase-exoglucanase pair from Trichoderma reesei.

The endoglucanase I (EGI) and the cellobiohydrolase I (CBHI) of the filamentous fungus Trichoderma reesei form a homologous pair of cellulolytic enzymes which nevertheless have different modes of action. We show here that the action of CBHI on bacterial microcrystalline cellulose results in efficient solubilisation but only a slow decrease in its degree of polymerisation. In contrast, the action of EGI results in a rapid decrease of the degree of polymerisation but less efficient overall solubilisation of the substrate. CBHI alone was practically inactive toward cotton which has a high initial degree of polymerisation and a complex morphology. EGI rapidly reduced the degree of polymerisation of cotton, and slowly solubilised part of it. Working synergistically, EGI and CBHI solubilised cotton more rapidly and to a greater extent than EGI alone. Our data are consistent with the exoglucanase nature of CBHI and also provide some evidence supporting its processive mode of action.

Bacteria↗
PubMed1998

A yeast t-SNARE involved in endocytosis.

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.

Amino Acid Sequence↗
PubMed1998

The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot.

The enamel knot, a transient epithelial structure, appears at the onset of mammalian tooth shape development. Until now, the morphological, cellular and molecular events leading to the formation and disappearance of the enamel knot have not been described. Here we report that the cessation of cell proliferation in the enamel knot in mouse molar teeth is linked with the expression of the cyclin-dependent kinase inhibitor p21. We show that p21 expression is induced by bone morphogenetic protein 4 (BMP-4) in isolated dental epithelia. As Bmp-4 is expressed only in the underlying dental mesenchyme at the onset of the enamel knot formation, these results support the role of the cyclin-dependent kinase inhibitors as inducible cell differentiation factors in epithelial-mesenchymal interactions. Furthermore, we show that the expression of p21 in the enamel knot is followed by Bmp-4 expression, and subsequently by apoptosis of the differentiated enamel knot cells. Three-dimensional reconstructions of serial sections after in situ hybridization and Tunel-staining indicated an exact codistribution of Bmp-4 transcripts and apoptotic cells. Apoptosis was stimulated by BMP-4 in isolated dental epithelia, but only in one third of the explants. We conclude that Bmp-4 may be involved both in the induction of the epithelial enamel knot, as a mesenchymal inducer of epithelial cyclin-dependent kinase inhibitors, and later in the termination of the enamel knot signaling functions by participating in the regulation of programmed cell death. These results show that the life history of the enamel knot is intimately linked to the initiation of tooth shape development and support the role of the enamel knot as an embryonic signaling center.

Animals↗
PubMed1997

Mso1p: a yeast protein that functions in secretion and interacts physically and genetically with Sec1p.

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1-1 mutation. We now describe a third suppressor of sec1-1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

Biological Transport↗
PubMed1997

Immunocytochemical analysis of Uukuniemi virus budding compartments: role of the intermediate compartment and the Golgi stack in virus maturation.

Previous studies have suggested that Uukuniemi virus, a bunyavirus, matures at the membranes of the Golgi complex. In this study we have employed immunocytochemical techniques to analyze in detail the budding compartment(s) of the virus. Electron microscopy of infected BHK-21 cells showed that virus particles are found in the cisternae throughout the Golgi stack. Within the cisternae, the virus particles were located preferentially in the dilated rims. This would suggest that virus budding may begin at or before the cis Golgi membranes. The virus budding compartment was studied further by immunoelectron microscopy with a pre-Golgi intermediate compartment marker, p58, and a Golgi stack marker protein, mannosidase II (ManII). Virus particles and budding virus were detected in ManII-positive Golgi stack membranes and, interestingly, in both juxtanuclear and peripheral p58-positive elements of the intermediate compartment. In cells incubated at 15 degrees C the nucleocapsid and virus envelope proteins were seen to accumulate in the intermediate compartment. Immunoelectron microscopy demonstrated that at 15 degrees C the nucleocapsid is associated with membranes that show a characteristic distribution and tubulo-vesicular morphology of the pre-Golgi intermediate compartment. These membranes contained virus particles in the lumen. The results indicate that the first site of formation of Uukuniemi virus particles is the pre-Golgi intermediate compartment and that virus budding continues in the Golgi stack. The results raise questions about the intracellular transport pathway of the virus particles, which are 100 to 120 nm in diameter and are therefore too large to be transported in the 60-nm-diameter vesicles postulated to function in the intra-Golgi transport. The distribution of the virus in the Golgi stack may imply that the cisternae themselves have a role in the vectorial transport of virus particles.

Animals↗
PubMed1996

Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei.

Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter. An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of beta-galactosidase expression of a large number of transformants. A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter. Removal of sequences upstream of nucleotide -500 in relation to the initiator ATG abolished glucose repression. Mutation of a single hexanucleotide sequence 5'GTGGGG at nucleotide -720 was sufficient for derepression. This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi. Removal of the glucose repressor site did not affect sophorose induction. Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position -161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box.

Base Sequence↗
PubMed1996

Use of a modified alcohol dehydrogenase, ADH1, promoter in construction of diacetyl non-producing brewer's yeast.

The bacterial gene, encoding alpha-acetolactate decarboxylase (alpha-ALDC), was expressed in a bottom-fermenting brewer's yeast under the control of a modified Saccharomyces cerevisiae alcohol dehydrogenase (ADH1) promoter which lacks the upstream regions from -800 bp to -1500 bp. In pilot scale brewing conditions, the level of alpha-ALDC produced was high enough to reduce the concentration of diacetyl so that lagering was not required. alpha-ALDC active brewer's yeast strains were also shown to be suitable for high gravity brewing.

Alcohol Dehydrogenase↗