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K Esser

Publications and source records attributed to K Esser.

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

Autocrine phosphorylation of p70(S6k) in response to acute stretch in myotubes.

Phosphorylation of 70-KDa S6 kinase (p70(S6k)) is correlated with in vivo skeletal muscle hypertrophy. Experiments tested whether mechanical stretch is sufficient to increase p70(S6k) phosphorylation in skeletal myotubes. Immediately following stretch, there was a small increase in p70(S6k) phosphorylation (63.2 +/- 8.5%) with maximal phosphorylation at 3 h (129.5 +/- 22.2%) and it remained elevated through 24 h (46.0 +/- 17.2%). To test whether an autocrine mechanism is involved, unstretched myotubes were incubated with medium from the stretch group for 10 min. Conditioned medium resulted in the phosphorylation of p70(S6k) in unstretched myotubes (92.8 +/- 28.9%) to levels comparable to the 3-h stretch group. These data indicate that p70(S6k) is phosphorylated in stretched myotubes via a mechanism that most likely involves an autocrine signaling pathway.

Animals↗
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↗
PubMed1999

The CACC box and myocyte enhancer factor-2 sites within the myosin light chain 2 slow promoter cooperate in regulating nerve-specific transcription in skeletal muscle.

Previous experiments showed that activity of the -800-base pair MLC2slow promoter was 75-fold higher in the innervated soleus (SOL) compared with the noninnervated SOL muscles. Using in vivo DNA injection of MLC2slow promoter-luciferase constructs, the aim of this project was to identify regulatory sites and potential transcription factors important for slow nerve-dependent gene expression. Three sites within the proximal promoter (myocyte enhancer factor-2 (MEF2), E-box, and CACC box) were individually mutated, and the effect on luciferase expression was determined. There was no change in luciferase expression in the SOL and extensor digitorum longus (EDL) muscles when the E-box was mutated. In contrast, the MEF2 mutation resulted in a 30-fold decrease in expression in the innervated SOL muscles (10.3 versus 0.36 normalized relative light units (RLUs)). Transactivation of the MLC2slow promoter by overexpressing MEF2 was only seen in the innervated SOL (676,340 versus 2,225,957 RLUs; p < 0.01) with no effect in noninnervated SOL or EDL muscles. These findings suggest that the active MLC2slow promoter is sensitive to MEF2 levels, but MEF2 levels alone do not determine nerve-dependent expression. Mutation of the CACC box resulted in a significant up-regulation in the EDL muscles (0.23 versus 4.08 normalized RLUs). With the CACC box mutated, overexpression of MEF2 was sufficient to transactivate the MLC2slow promoter in noninnervated SOL muscles (27,536 versus 1, 605,797 RLUs). Results from electrophoretic mobility shift and supershift assays confirm MEF2 protein binding to the MEF2 site and demonstrate specific binding to the CACC sequence. These results suggest a model for nerve-dependent regulation of the MLC2slow promoter in which derepression occurs through the CACC box followed by quantitative expression through enhanced MEF2 activation.

Animals↗
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↗
PubMed1999

Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise.

High-resistance exercise training results in an increase in muscle wet mass and protein content. To begin to address the acute changes following a single bout of high-resistance exercise, a new model has been developed. Training rats twice a week for 6 wk resulted in 13.9 and 14.4% hypertrophy in the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, respectively. Polysome profiles after high-resistance lengthening contractions suggest that the rate of initiation is increased. The activity of the 70-kDa S6 protein kinase (p70(S6k)), a regulator of translation initiation, is also increased following high-resistance lengthening contractions (TA, 363 +/- 29%; EDL, 353 +/- 39%). Furthermore, the increase in p70(S6k) activity 6 h after exercise correlates with the percent change in muscle mass after 6 wk of training (r = 0.998). The tight correlation between the activation of p70(S6k) and the long-term increase in muscle mass suggests that p70(S6k) phosphorylation may be a good marker for the phenotypic changes that characterize muscle hypertrophy and may play a role in load-induced skeletal muscle growth.

Animals↗
PubMed1999

Transcriptional regulation in response to exercise.

Much progress has been made in recent years into understanding molecular mechanisms by which transcription is regulated following changes in physiological stimuli. This review has tried to focus on what is known about four specific physiological challenges--mechanical load, intracellular calcium, hypoxia, and redox state. Because of our biased interest in exercise, it was our goal to review these relatively well-studied systems so that we might provide insight into potential mechanisms that govern exercise-induced transcriptional changes. What becomes obvious, when reaching the end of this review, is that there are many common themes among the different physiological responses described. Some examples include the activation of IEGs, such as c-jun and c-fos, the phosphorylation of the transcription factor CREB, and the importance of the serum response element and the serum response factor. These commonalities across the different physiological systems suggest a certain redundancy or shared mechanism(s) for regulating transcription in response to physiological stimuli. While very little is known at this time about how exercise regulates transcription, it is an exciting time in this field of research. The recent growth in the molecular biological research literature of more physiologically-based studies provides exciting new molecular and cellular tools for those researchers willing to take on the challenge of understanding the complex mechanisms of exercise-induced adaptations.

Calcium↗
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

Exercise down-regulates hepatic lipogenic enzymes in food-deprived and refed rats.

The effects of an acute bout of prolonged exercise on the activities of several hepatic lipogenic enzymes and the abundance of fatty acid synthase (FAS) mRNA were evaluated using a food deprivation-refeeding protocol in which diets contained 50% of the energy from either fructose or cornstarch. Food was withheld from male rats for 48 h and refed for 0, 4, 8, 12, 24 or 48 h. At each time point, half of each dietary group was subjected to a single bout of treadmill running until exhaustion and killed immediately. The other half of each group rested without food for the same amount of time before being killed. Exercise significantly decreased FAS activity by 57, 46, 10, 26 and 70% at 4, 8, 12, 24 and 48 h of refeeding, respectively, in the fructose-fed rats; and by 70 and 63% at 24 and 48 h of refeeding, respectively, in the cornstarch-fed rats. Activities of L-type pyruvate kinase and glucose 6-phosphate dehydrogenase were significantly decreased after exercise in the fructose-fed, but not cornstarch-fed rats. In rested rats, FAS mRNA abundance increased approximately fourfold above the unfed levels after 8 and 12 h of refeeding. Exercise attenuated the diet-induced increases in FAS mRNA abundance. At 8 h of refeeding, both cornstarch- and fructose-fed exercised rats had 71% (P < 0.05) of the FAS mRNA levels of their rested counterparts; at 12 h, these exercised rats showed only 46 and 27% (P < 0.05) of FAS mRNA levels compared with rested rats fed the same diet. We conclude that dietary induction of FAS activity and mRNA abundance can be inhibited by prolonged exercise, suggesting that exercise may influence FAS transcription and/or mRNA stability.

Animals↗
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↗
PubMed1993

Nerve-dependent and -independent patterns of mRNA expression in regenerating skeletal muscle.

Muscle regeneration was used to assess the roles of innervation and intrinsic genetic information in the acquisition of predominant slow and fast contractile protein mRNA profiles in adult skeletal muscle. Rat soleus (slow) and EDL (fast) muscles were allowed to regenerate in the presence and absence of their respective slow and fast nerves. Levels of mRNAs encoding fast, slow, and cardiac contractile protein isoforms were quantitatively determined at specific times during regeneration. All muscles initially expressed a heterogeneous pattern of fast, slow, and cardiac isoform mRNAs during myotube formation. Subsequently, the EDL muscle made a transition to a predominantly fast profile of mRNAs which was independent of innervation. The mRNA profile for the noninnervated regenerating soleus muscle was similar to both the innervated and the noninnervated EDL muscle profile. Thus, the decision to express predominantly fast isoform mRNAs is intrinsic to in vivo muscle regeneration with the fast nerve not appearing to be informative. In contrast, acquisition of a slow mRNA profile is dependent on the presence of a slow nerve. The mRNAs encoding slow isoforms from all of the contractile protein gene families are upregulated during the period of reestablishment of neuromuscular transmission. Additionally, there is no concomitant down-regulation of the fast isoform mRNAs upon reinnervation in the soleus regenerate. We propose that information both intrinsic to the muscle and supplied by the in vivo environment acts to provide potential isoform mRNA options to the regenerating muscle.

Animals↗
PubMed1993

Adjuvant arthritic (AA) rats exhibit enhanced endotoxin-induced plasma TNF (EIPT) levels.

Adjuvant arthritis (AA) was induced in male Lewis rats by a single FCA (M. butyricum) injection into the tail. At various time periods post FCA injection, AA and control rats were anesthetized and administered E. coli endotoxin (30 mg/kg, i.v.). Plasma samples were obtained at 30, 60, 90, 120 and 150 min following endotoxin administration and assayed for TNF alpha levels by ELISA. Compared to control rats, AA rats exhibited enhanced EIPT levels (706% of control, p < 0.05) which was associated with the onset of inflammatory lesions on days 12-14 post FCA, and remained significantly elevated (> 300% of control, p > 0.05) for at least 30 days post FCA. There were no significant correlations between EIPT levels and hindpaw volumes or body weights. The results of this study support previous observations that AA is associated with macrophage activation and suggest that EIPT levels in AA rats may be a useful parameter for the evaluation of novel antiarthritic agents.

Animals↗
PubMed1991

Molecular cloning and characterization of the recA gene of Methylomonas clara and construction of recA deficient mutant.

The recA gene of the methylotrophic bacterium Methylomonas clara has been isolated from a genomic library by hybridization with the Escherichia coli recA gene. Its complete nucleotide sequence consists of 1029 bp encoding a polypeptide of 342 amino acids. Nucleotide sequence analysis of the M. clara recA gene revealed extensive homologies to recA genes from E. coli and Pseudomonas aeruginosa. Part of the physiological activity of the M. clara RecA protein has become evident in that E. coli recA mutant HB101 is complemented. The cloned recA gene has been modified in vitro by site-specific mutagenesis and by insertion of a kanamycin-resistance gene cassette into the recA coding sequence. M. clara recA mutants were obtained by replacement of the active recA gene by an in-vitro inactivated gene copy.

Amino Acid Sequence↗
PubMed1991

Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes.

Protoplasts of wild-type strain s and a long-lived extrachromosomal mutant (AL2) of the ascomycete Podospora anserina were transformed using a plasmid (pAN7-1) which contains the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of Aspergillus nidulans regulatory sequences. After optimizing the transformation procedure, transformation efficiencies of 15-21 transformants/micrograms plasmid DNA were obtained. Using a second selectable vector (pBT3), which contains the beta-tubuline gene of a benomyl-resistant Neurospora crassa mutant, the co-transformation rate was determined. Southern blot hybridization experiments revealed that the transforming plasmid became integrated into the genome of the recipient either as a single copy or as multiple copies. In addition, the data from molecular as well as from classical genetic analyses indicated that in independent transformants vector integration occurred at different positions. The mitotic and meiotic stability of transformants proved to be dependent on the number of integrated plasmid copies. Genetic analyses revealed a transformant in which the integrated vector is closely linked to the mating-type locus. Fractionation of whole chromosomes by pulsed field gel electrophoresis and subsequent hybridization of the immobilized DNAs against radiolabelled vector sequences indicated the largest of seven chromosomes as the chromosome containing the integrated vector and thus the mating-type locus.

Ascomycota↗
PubMed1991

Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis.

Linear hybrid plasmids based on the killer plasmid pGKL1 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae. Like pGKL1, the hybrids are located in the cytoplasm, have terminal inverted repeats (TIR) and possess covalently linked proteins at their 5' ends. The construction of cytoplasmic hybrid plasmids is based on the use of a pGKL1 promoter to control the marker gene used for recombination. Nuclear promoters are not recognised in the cytoplasm.

Base Sequence↗
PubMed1991

Antiserum to a novel peptide sequence reacts selectively with epithelial subpopulations.

A synthetic peptide corresponding to a novel protein sequence isolated from bovine kidney was used to immunize rabbits. When applied to Western blots of bovine kidney extracts, antiserum to this peptide recognizes proteins with molecular weights of 23 and 18 KD. Immunohistochemical examination of a variety of bovine and rat tissues with this antiserum revealed a unique distribution of immunoreactivity with the intermediate layers of a variety of stratified epithelia, in addition to renal glomeruli. The pattern of reactivity differed from previously described epithelial markers such as cytokeratins. These results indicate that this antiserum may be useful as a tool for the identification of cells of the intermediate layer of stratified epithelia and, as such, may aid in the study of this differentiating/proliferating tissue compartment.

Amino Acid Sequence↗