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G Michaelis

Publications and source records attributed to G Michaelis.

At least 19 recordsLinked to original sources
PubMed2004

The mitochondrial IMP peptidase of yeast: functional analysis of domains and identification of Gut2 as a new natural substrate.

The mitochondrial inner membrane peptidase IMP of Saccharomyces cerevisiae is required for proteolytic processing of certain mitochondrially and nucleus-encoded proteins during their export from the matrix into the inner membrane or the intermembrane space. The membrane-associated signal peptidase complex is composed of the two catalytic subunits, Imp1 and Imp2, and the Som1 protein. The IMP subunits are thought to function in membrane association, interaction and stabilisation of subunits, substrate specificity, and proteolysis. We have analysed inner membrane peptidase mutants and substrates to gain more insight into the functions of various domains and investigate the basis of substrate recognition. The results suggest that certain conserved glycine residues in the second and third conserved regions of Imp1 and Imp2 are important for stabilisation of the Imp complex and for the proteolytic activity of the subunits, respectively. The non-conserved C-terminal parts of the Imp subunits are important for their proteolytic activities. The C-terminal region of Imp2, comprising a predicted second transmembrane segment, is dispensable for the stability of Imp2 and Imp1, and cannot functionally substitute for the C-terminal segment of Imp1. Alteration of the Imp2 cleavage site in cytochrome c(1) (from A/M to N/D) reveals the specificity of the Imp2 peptidase. In addition, we have identified Gut2, the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase, as a new substrate for Imp1. Failure to cleave the Gut2 precursor may contribute to the pet phenotype of certain imp mutants. Gut2 is associated with the inner membrane, and is essential for growth on glycerol-containing medium. Suggested functions of the analysed residues and domains of the IMP subunits, characteristics of the cleavage sites of substrates and implications for the phenotypes of imp mutants are discussed.

Amino Acid Sequence↗
PubMed2000

A screen of yeast respiratory mutants for sensitivity against the mycotoxin citrinin identifies the vacuolar ATPase as an essential factor for the toxicity mechanism.

In countries with a hot climate the mycotoxin citrinin represents a serious problem in fungal food-poisoning. In humans the renal system is affected the most and the mitochondrial respiratory chain was identified as a possible sensitive target for this toxin. In addition, citrinin has an antifungal activity that also inhibits the growth of the yeast Saccharomyces cerevisiae. So far the precise mode of action and the subcellular targets for citrinin have not been identified. Therefore, we decided to use the model organism yeast for a genetic approach to identify genes that play a role in the sensitivity against this mycotoxin. A large collection of conditional respiratory deficient yeast mutants was screened for sensitivity against citrinin. One special pet-ts mutant was identified that exhibited a higher sensitivity against citrinin. The genetic system of yeast allowed the isolation of the respective wild-type gene. This yeast gene encodes the Vph2p subunit that is essential for the correct assembly of the vacuolar ATPase. Isolation of the mutated gene and gene-disruption experiments of VPH2 and the partially overlapping small YKL118W gene verified this finding. The wild-type VPH2 gene restores all defects of the mutants. In contrast to this, YKL118W gave no complementation and the null mutant showed no phenotype. Thereby the yeast vacuolar ATPase was found to be important for the toxic effect of citrinin in yeast cells. The consequences of this finding for the molecular mechanism of citrinin action and its relation to the mitochondrial respiratory chain are discussed.

Adenosine Triphosphatases↗
PubMed2000

Som1, a third component of the yeast mitochondrial inner membrane peptidase complex that contains Imp1 and Imp2.

The mitochondrial inner membrane peptidase Imp is required for proteolytic processing of the mitochondrially encoded protein Cox2, the nucleus-encoded Cyt b2, Mcr1, and Cyt c1, and possibly other proteins, during their transport across the mitochondrial membranes. The peptidase contains two catalytic subunits, Imp1 and Imp2. The small protein Soml was previously shown to affect the function of Imp1, but the precise role of Soml remained unknown. Using mutants deleted for IMP1, IMP2 and SOM1, we show here that the Som1 protein is absent in the imp1delta mutant, whereas the level of the Imp1 subunit of the peptidase is only slightly reduced in the soml null mutant. The Soml protein is not essential for proteolytic processing of Cyt b2, while the two other known Imp1 substrates, Cox2 and Mcr1, are not processed in the absence of Som1. Proteolytic processing of Cyt c1 by the Imp2 subunit, and of Ccp by an as yet unidentified peptidase, is not impaired in the som1 deletion mutant. By crosslinking and co-immunoprecipitation assays we demonstrate that the Imp1 and Som1 proteins physically interact. We conclude from our results that stabilisation of Som1 and correct Imp1 function is mediated by a direct interaction between the Imp1 and Som1 proteins, suggesting that Som1 represents a third subunit of the Imp peptidase complex.

Cross-Linking Reagents↗
PubMed2000

[Clinical significance and effects of foreign body embolism during the use of central venous catheters].

An embolism caused by catheters or puncture devices is generally a rare complication during the use of central venous catheters. Possible reasons are either mistakes on the part of the user (e.g. shearing off or tearing of catheters or Seldinger wires) or other accidental causes (e.g. faulty products, material fatigue). As patients are at risk of suffering serious injuries following an embolism, foreign bodies should be removed from the patient's cardio circulatory system as quickly and as completely as possible. Two-dimensional echo-cardiography has proved to be very helpful for diagnostics, while interventional radiology is most effective for the removal of intravascular foreign bodies.

Catheterization, Central Venous↗
PubMed1999

Disruption of six open reading frames on chromosome X of Saccharomyces cerevisiae reveals a cluster of four essential genes.

In this study we report the construction and basic phenotypic analysis of six Saccharomyces cerevisiae deletion mutants. The open reading frames (ORFs) YJL008C (gene symbol CCT8), YJL010C, YJL011C, YJL012C, YJL017W, and YJL020C from chromosome X have been disrupted by integration of deletion cassettes, comprising the bacterial KanMX4 marker gene and terminal long (LFH) or short (SFH) flanking sequences that are homologous to the 5' and 3' untranslated regions of the respective ORFs. For correct disruption of ORF YJL008C, it was necessary to construct a deletion cassette flanked by 300-350 bp long target guide sequences by LFH-PCR. Transformations using ORF YJL008C gene disruption cassettes synthesized by standard SFH-PCR exclusively resulted in false-positive or multiple integration events, probably because seven additional genes homologous to CCT8 exist in the yeast genome. The other five ORFs have been disrupted using cassettes generated by SFH-PCR, comprising terminal homologous regions of approximately 50 bp to each target site. Correct genomic integration of the reporter modules was verified by analytical PCR and Southern hybridization. Deletion of YJL008C, YJL010C, YJL011C, and YJL012C was found to be lethal, as shown by sporulation and tetrad analysis. This result is in contrast to the finding that only 16-20% of the genes in S. cerevisiae are estimated to be essential. The four essential genes described in this work are clustered, while the two other non-essential ORFs are separated by further ORFs. Although the two viable deletion mutants were tested against 60 different inhibitors, heavy metal ions and salts, no phenotype could be detected that co-segregated with the deletion during meiosis.

Amino Acid Sequence↗
PubMed1998

The Saccharomyces cerevisiae SOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane.

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28 degrees C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S. cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane.

Amino Acid Sequence↗
PubMed1996

SOM 1, a small new gene required for mitochondrial inner membrane peptidase function in Saccharomyces cerevisiae.

IMP1 encodes a subunit of the mitochondrial inner membrane peptidase responsible for the proteolytic processing of cytochrome oxidase subunit 2 (Cox2) and cytochrome b2 (Cytb2). The molecular defect in an imp1 mutation and the characterisation of a high-copy-number suppressor is described. A deletion of the suppressor region causes respiration deficiency. The DNA sequence revealed three very small overlapping ORFs. Constructs which carried termination codons within the ORFs or lacked ATG initiation codons still retained complementing activity on a high-copy-number plasmid. Nevertheless, the possibility that the suppressor acts at DNA or RNA level could be excluded. Subcloning of the ORFs, complementation analysis in low-copy-number plasmids and transcript mapping identified the 222 bp ORF as the suppressor gene designated SOM1. The SOM1 gene is transcribed into a 375 bp polyadenylated RNA and the deduced amino acid sequence predicts a small protein of 8.4 kDa with no significant sequence similarity to known proteins. In the som1 deletion mutant, proteolytic processing of the Cox2 precursor is prevented and Cytb2 is strongly reduced. SOM1 represents a new small gene which encodes a novel factor that is essential for the correct function of the Imp1 peptidase and/or the protein sorting machinery.

Amino Acid Sequence↗
PubMed1996

A new point mutation in the nuclear gene of yeast mitochondrial RNA polymerase, RPO41, identifies a functionally important amino-acid residue in a protein region conserved among mitochondrial core enzymes.

The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6. In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria. In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase. A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme. Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant. The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine. This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases. However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.

Amino Acid Sequence↗
PubMed1995

[Mechanical autologous transfusion in orthopedic surgery in children. Is the use of mechanical autologous transfusion possible even in pediatric orthopedic surgical procedures?].

The use of autotransfusion devices is an established method of reducing the need for homologous transfusions in surgery [3, 11, 13], but technical factors still contraindicate the washing and concentration of blood volumes smaller than 300 ml. Therefore, haemoconcentration of small volumes of salvaged blood, as usually found in paediatric surgery, is considered to be a complicated and questionable practice [5]. Whereas these amounts of blood loss are easily tolerated by adults, they may necessitate homologous transfusions in paediatric surgery. In a prospective study, we investigated whether a simple technical modification in the processing of salvaged blood could facilitate the use of autotransfusion devices, especially in children. PATIENTS AND METHODS. Intraoperative blood salvage was performed in children 6 months to 10 years old undergoing surgery for hip dysplasia. Autotransfusion (Dideco STAT) was started when the blood loss was estimated to be more than 20% of the total blood volume (TBV). As a reference, we used a formula based on body weight [10]: for children up to the age of 6 years 80 ml/kg blood volume and for children up to 10 years 75 ml/kg. The total volume of salvaged fluid including blood, anticoagulant solution, and surgical irrigation was collected in a reservoir and transferred to the autotransfusion set, after which the reservoir was rinsed with 500 ml 0.9% saline solution in order to save the remaining blood. After processing, the blood was stored in the retransfusion bag. By adding the same volume of plasma expander (6% hydroxyethyl starch [HES], molecular weight 450,000), spontaneous sedimentation of the washed autologous erythrocytes (RBCs) for 10-15 min led to a concentrate of RBCs. After 10 mu filtration, the RBC suspension was retransfused (Figs. 1-3). RESULTS. Within 12 months, autotransfusion was performed during 6 out of 15 surgical procedures according to the method described above. The calculated blood loss averaged 25.6% of TBV, of which 21.4% (= 272 ml) could be processed by the autotransfusion device (Table 3). The mean values of 2.6 g/dl haemoglobin (Hb) and 6.8% haematocrit (HCt) in the salvaged blood increased to 9.4 g/dl and 27.3% in the processed RBC concentrates. After adding 6% HES solution, spontaneous sedimentation of the RBCs led to values of Hb 22.1 g/dl and HCt 59.8%. An average of 59.5 ml (22-99 ml) sedimented RBCs was retransfused to the patients, including 11.6 ml 6% HES solution (Table 4). In this manner, the need for homologous transfusions could be avoided in these patients both during and after surgery. CONCLUSIONS. This study shows that the use of blood salvaging in paediatric surgery is indicated under certain conditions. With the aid of the simple modification described above, we solved the main problem in paediatric autotransfusion by concentrating RBC suspensions with low Hb and Hct values after using the autotransfusion device.

Blood Sedimentation↗
PubMed1994

PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast.

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 bp upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.

Alleles↗
PubMed1994

A new nuclear suppressor system for a mitochondrial RNA polymerase mutant identifies an unusual zinc-finger protein and a polyglutamine domain protein in Saccharomyces cerevisiae.

A yeast strain with a point mutation in the nuclear gene for the core subunit of mitochondrial RNA polymerase was used to isolate new extragenic suppressors. Spontaneously occurring phenotypical revertants were analysed by crosses with the wild-type and tetrad dissection. One of the new nuclear suppressor mutants was characterized by temperature-sensitive growth on non-fermentable carbon sources. This mutant was transformed with a genomic yeast library. Two independent types of DNA clones were isolated which both complemented the temperature-sensitive defect. Subcloning and DNA sequencing identified two novel yeast genes which code for proteins with the characteristic features of transcription factors. Both factors exhibit highly structured protein domains consisting of runs and clusters of asparagine and glutamine residues. One of the proteins contains in addition zinc-finger domains of the C2H2-type. Therefore the genes are proposed to be named AZF1 (asparagine-rich zinc-finger protein) and PGD1 (polyglutamine domain protein). Gene disruption of both reading frames has no detectable influence on the vegetative growth on complete glucose or glycerol media, indicating that the genes may act as high copy number suppressors of the mutant defect. Additional transformation experiments showed that AZF1 is also an efficient suppressor for the original defect in the core subunit of mitochondrial RNA polymerase.

Amino Acid Sequence↗
PubMed1993

Nuclear control of the messenger RNA expression for mitochondrial ATPase subunit 9 in a new yeast mutant.

A new temperature-sensitive nuclear mutant of Saccharomyces cerevisiae altered in the expression of the mitochondrially encoded ATPase 9 gene was identified. Analysis of several mitochondrial transcripts reveals the selective loss of the messenger RNA for the ATPase 9 subunit under restrictive temperature conditions. This defect results in a respiratory deficiency of the yeast cells. RNA synthesis studied with isolated mitochondria gives first evidence that the mitochondrial transcription process does not react in a temperature-sensitive way. Therefore, it is concluded that the loss of the transcript is due to specific posttranscriptional degradation. Further on it is demonstrated that all the observed effects in the mutant are caused by only one mutated nuclear gene. The mutant strain was transformed with a genomic yeast library and a complementary DNA fragment was obtained. Molecular characterization of the respective DNA clone identified a new gene, which we propose to call NCA1 (Nuclear Control of ATPase messenger RNA expression). Complete inactivation of this gene by insertion mutagenesis in the chromosome leads to a permanent respiratory defect of the cell. Localization of the insertion locus in the yeast genome and complementation studies with the temperature-sensitive mutant indicate that the two mutations are allelic.

Alleles↗
PubMed1993

A point mutation in the core subunit gene of yeast mitochondrial RNA polymerase is suppressed by a high level of specificity factor MTF1.

The temperature-sensitive yeast mutant pet-ts798 is characterized by an altered mitochondrial transcription apparatus. The mutation has previously been shown to map in the RPO41 gene encoding the core enzyme of mitochondrial RNA polymerase. In the present study the rpo41/pet-ts798 allele was cloned and sequenced, demonstrating that the mutant phenotype is caused by a single amino acid change in a conserved region of the core polymerase. The nuclear gene MTF1, previously isolated as a high copy suppressor of mutant rpo41/pet-ts798, and its gene product were characterized in more detail. Import of a MTF1-COXIV fusion protein in vivo and also import studies with in vitro synthesized MTF1 precursors indicate that MTF1 is a mitochondrial protein and that no apparent cleavage occurs during its import into mitochondria. DNA-binding assays demonstrate that the MTF1 protein alone interacts with DNA in a non-specific manner. An antibody directed against specificity factor MTF1 was raised and used for immunological quantification experiments. The results indicate that suppression is mediated by an increased level of MTF1 protein in mitochondria of the rpo41/pet-ts798 mutant. Possible implications of this finding for the mechanism of suppression are discussed.

Alleles↗
PubMed1993

Mitochondrial DNA of Chlamydomonas reinhardtii: the structure of the ends of the linear 15.8-kb genome suggests mechanisms for DNA replication.

The mitochondrial genome of Chlamydomonas reinhardtii is a linear double-stranded DNA of 15.8 kb. With the exception of the termini its DNA sequence has been published. Here we describe the unique structure of the two termini determined from cloned fragments or, for the very terminal sequences, by the Maxam and Gilbert method after 5' labeling of uncloned terminal fragments. The 15.8-kb DNA is characterized by terminal inverted repeats of 531 or 532 bp in length including long 3' extensions. The 3' single-stranded extensions of the left and right ends are non-complementary, identical in sequence, and comprise 39 to 41 nucleotides. Remarkably, the linear genome possesses in addition an internal 86-bp repeat of the two outermost sequences. The unusual structure of the 15.8-kb DNA termini is compared with those of other linear mitochondrial DNAs. Possible mechanisms of 15.8-kb DNA replication are discussed.

Animals↗
PubMed1991

Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast.

Several precursors transported from the cytoplasm to the intermembrane space of yeast mitochondria are first cleaved by the MAS-encoded protease in the matrix space and then by additional proteases that have not been characterized. We have now developed a specific assay for one of these other proteases. The enzyme is an integral protein of the inner membrane; it requires divalent cations and acidic phospholipid for activity, and is defective in yeast mutant pet ts2858 which accumulates an incompletely processed cytochrome b2 precursor. The protease contains a 21.4 kd subunit whose C-terminal part is exposed on the outer face of the inner membrane. An antibody against this polypeptide inhibits the activity of the protease. As overproduction of the polypeptide does not increase the activity of the protease in mitochondria, the enzyme may be a hetero-oligomer. This 'inner membrane protease I' shares several key features with the leader peptidase of Escherichia coli and the signal peptidase of the endoplasmic reticulum.

Amino Acid Sequence↗