Wolfhard Bandlow

A simple method for the large-scale preparation of mitochondria from microorganisms

Abstract A simple mechanical procedure that has been developed for the large-scale preparation of intact mitochondria from yeast, is also applicable to the extraction of organelles from other organisms having cell walls. A procedure for the isolation of large quantities of pure mitochondrial DNA from these mitochondria is described. In Schizosaccharomyces pombe, further purification of the mitochondria by urografin isopycnic centrifugation leads to 50% recovery of whole cell respiration activity in a vesicular fraction of respiratory chain enzymes, with NADH oxidase activity usually greater than 10 μmol of electrons/min/mg of protein. The method has the advantage of rapidity and low cost and it is extremely healthy for the operator.

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). Recombinant Rio1p from Escherichia coli and tagged Rio1p from yeast has kinase activity in vitro, and mutation of amino acid residues that are conserved and indispensable for catalytic activity (i.e. ATP‐binding motif, catalytic centre) abrogates activity. RIO1 is essential in yeast and plays a role in cell cycle progression. After sporulation of RIO1/rio1 diploids, RIO1‐disrupted progeny cease growth after one to three cell divisions and arrest as either large unbudded or large‐budded cells. Cells deprived of Rio1p are enlarged and arrest either in G1 or in mitosis mainly with the DNA at the bud neck and short spindles (a phenotype also seen in cells carrying a weak allele), suggesting that Rio1p activity is required for at least at two steps during the cell division cycle: for entrance into S phase and for exit from mitosis. The weak RIO1 allele leads to increased plasmid loss.

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, PFYI, encoding an actin‐binding protein, leads to no very obvious phenotype. Interestingly, profilin over‐production 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.

Membrane separation and biogenesis of the outer membrane of yeast mitochondria

1. 1. Isotonic swelling and shrinking of the mitochondria of Saccharomyces cerevisiae in the presence of valinomycin and subsequent sucrose gradient centrifugation resulted in the separation of the inner and outer mitochondrial membranes. Succinate: cytochrome c reductase and malate dehydrogenase served as markers for the inner membrane and the matrix, respectively, adenylate kinase for the intermembrane space. 2. 2. The outer membrane fraction is characterized by the activities of both antimycin A-insensitive NADH: cytochrome c reductase and kynurenine hydroxylase. The latter is shown to be exclusively bound to the outer mitochondrial membrane in yeast. Kynurenine hydroxylase exhibits a sharp pH optimum at about pH 7.4. Yeast mitochondria fail to show monoamine oxidase activity. 3. 3. The products of mitochondrial and cytoplasmic protein synthesis in Saccharomyces cerevisiae were each selectively labelled in vivo. The fractions obtained after membrane separation were characterized by their 3H to 14C ratio. The outer membrane of yeast mitochondria is not pulse-labelled by radioactive leucine incorporation in the presence of cycloheximide and hence is apparently not synthesized by the mitochondial protein synthesizing system. More than one third of the inner membrane protein is synthesized on mitrochondrial ribosomes. 4. 4. As estimated from enrichment in enzyme activity and in specific radioactivity, the outer membrane fraction contains 6–8%, the inner about 30% of the total protein in yeast mitochondria. 5. 5. Intact mitochondria, outer and inner membranes exhibit densities of 1.173 g/cm3, 1.084 g/cm3 and 1.190 g/cm3, respectively 6. 6. All mitochondrial subfractions obtained after membrane separation were examined for their morphological appearance by electron microscopy.

The Yeast Trimeric Guanine Nucleotide-Binding Protein α Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients

ABSTRACT Saccharomyces cerevisiae Gpa2p, the α 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.

A nuclear yeast gene (GCY) encodes a polypeptide with high homology to a vertebrate eye lens protein

We describe here the nuclear gene for a yeast protein showing unexpectedly high homology with mammalian aldo/keto reductases as well as with ϱ‐crystallin, one of the prominent proteins of the frog eye lens. Although it could be proven that the gene occurs as a single copy in the haploid yeast genome, replacement of the intact by a disrupted, nonfunctional allele led to no obvious phenotype, indicating that the gene is dispensable. The gene was assigned to chromosome XV. It is transcribed in vivo into an mRNA of about 1300 bases with a coding capacity for a protein of 312 amino acids (estimated M r 35000).

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. An initial biochemical characterization of Rio1p is presented.

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. ThePFY1 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·dT element. Reb1p binds specificallyin vitro. Mutation of this site reduces PFY1expression to about 35%. A nucleosome-free gap of about 190 bp is centered at the genomic Reb1p binding site in vivo and spans the presumptive core promoter and transcriptional initiation sites. Nucleosomes at the border of the gap are positioned. Mutation of the Reb1p motif in the genomic PFY1 promoter abolishes nucleosome positioning, fills the gap with a non-positioned nucleosome, and reduces transcription by a factor of 3. From permutation studies we conclude that Reb1p induces a strong bend into the DNA. Phasing analyses indicate that it is directed toward the major groove. The data suggest that Reb1p plays an architectural role on DNA and that Reb1p-dependent DNA bending leads to a DNA conformation that is incompatible with packaging into nucleosomes and concomitantly facilitates constitutive transcription. In the absence of other transcription activators, Reb1p excludes nucleosomes and moderately stimulates transcription by distorting DNA.

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.

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.