The subcellular distribution of fumarase isozymes in rat liver

Between 13 and 25% of the fumarase activity of rat liver was found to be cytosolic in origin the remainder being localised in the mitochondria. Electrophoretic analysis on cellulose acetate showed that mitochondria do not contain detectable levels of cytosolic isozyme or vice versa.     

Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells

For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.     

A quantitative study of the subcellular distribution of aspartate aminotransferase isozymes in rat liver

• 1.1. Between 15 and 19% of the aspartate aminotransferase activity of rat liver is of cytosolic origin; the remainder is localised entirely in the mitochondria.
• 2.2. Mitochondria do not contain detectable levels of the cytosolic isozyme or vice versa.
• 3.3. In solutions of low ionic strength, damaged mitochondria release only small amounts of aspartate aminotransferase. but the enzyme is released quantitatively by exposure to high concentrations of salt.

     

Genome-wide analysis of integral membrane proteins from eubacterial,archaean, and eukaryotic organisms.

We have carried out detailed statistical analyses of integral membrane proteins of the helix-bundle class from eubacterial, archaean, and eukaryotic organisms for which genome-wide sequence data are available. Twenty to 30% of all ORFs are predicted to encode membrane proteins, with the larger genomes containing a higher fraction than the smaller ones. Although there is a general tendency that proteins with a smaller number of transmembrane segments are more prevalent than those with many, uni-cellular organisms appear to prefer proteins with 6 and 12 transmembrane segments, whereas Caenorhabditis elegans and Homo sapiens have a slight preference for proteins with seven transmembrane segments. In all organisms, there is a tendency that membrane proteins either have many transmembrane segments with short connecting loops or few transmembrane segments with large extra-membraneous domains. Membrane proteins from all organisms studied, except possibly the archaeon Methanococcus jannaschii, follow the so-called "positive-inside" rule; i.e., they tend to have a higher frequency of positively charged residues in cytoplasmic than in extra-cytoplasmic segments.     

Comparison of eubacterial and eukaryotic 5S RNA structures: a chemical modification study

The 5S RNAs from Bacillus stearothermophilus and Saccharomyces cerevisiae were probed by nucleotide-specific reagents, with a view to compare and contrast their higher order structures. The progressive unfolding of the RNAs during heating, in the presence and absence of magnesium, was monitored. Evidence was provided for the double-helical segments which occur in the secondary structural models of both RNAs. The results also placed constraints on the possible structuring of the remainder of the RNA and yielded some insight into ways of folding up the molecule. Together with the data from our earlier studies, employing ribonucleases, these results provide a detailed picture of the structuring and topography of the 5S RNAs. The main structural differences between the eubacterial and eukaryotic RNAs occur throughout the loop D/helix IV/loop E/helix V arm; in particular strong evidence is provided for loop D of the eukaryotic RNA being involved in a tertiary interaction.     

Comparison of eubacterial and eukaryotic 5S RNA structures: a Raman spectroscopic study

Raman spectra of eubacterial ribosomal 5S RNAs of Escherichia coli, Bacillus subtilis and Thermus thermophilis and of eukaryotic 5S RNAs of yeast and rat liver have been compared. The spectra show a very high and comparable regularity in the ribophosphate backbone as indicated by the ratio 1.67±0.03 for I₈₁₂/I₁₁₀₀ in all samples. The 5S RNAs studied have a similar degree of stacking of the G, A and pyrimidine bases. A high percentage of base-paired U residues between 43 and 66% is indicated. Conformational alterations occurring in 5S RNAs in the presence of Mg²⁺ ions between 20 and 50^*C are localized mainly in the region of loop II of the molecule. The implications of these results for the 5S RNA structure are discussed.     

Extreme zinc tolerance in acidophilic microorganisms from the bacterial and archaeal domains

Zinc can occur in extremely high concentrations in acidic, heavy metal polluted environments inhabited by acidophilic prokaryotes. Although these organisms are able to thrive in such severely contaminated ecosystems their resistance mechanisms have not been well studied. Bioinformatic analysis of a range of acidophilic bacterial and archaeal genomes identified homologues of several known zinc homeostasis systems. These included primary and secondary transporters, such as the primary heavy metal exporter ZntA and Nramp super-family secondary importer MntH. Three acidophilic model microorganisms, the archaeon ??Ferroplasma acidarmanus??, the Gram negative bacterium Acidithiobacillus caldus, and the Gram positive bacterium Acidimicrobium ferrooxidans, were selected for detailed analyses. Zinc speciation modeling of the growth media demonstrated that a large fraction of the free metal ion is complexed, potentially affecting its toxicity. Indeed, many of the putative zinc homeostasis genes were constitutively expressed and with the exception of ??F. acidarmanus?? ZntA, they were not up-regulated in the presence of excess zinc. Proteomic analysis revealed that zinc played a role in oxidative stress in At. caldus and Am. ferrooxidans. Furthermore, ??F. acidarmanus?? kept a constant level of intracellular zinc over all conditions tested whereas the intracellular levels increased with increasing zinc exposure in the remaining organisms.     

Protein domains, catalytic activity, and subcellular distribution of mouse NTE-related esterase

A mammalian family of lipid hydrolases, designated “patatin-like phospholipase domain containing (PNPLA)” recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.     

Differential subcellular targeting and activity-dependent subcellular localization of diacylglycerol kinase isozymes in transfected cells

Diacylglycerol kinase (DGK) plays a pivotal role in cellular signal transduction through regulating levels of the second messenger diacylglycerol (DG). Previous studies have revealed that DGK is composed of a family of isozymes that show remarkable heterogeneity in terms of molecular structure, functional domains, tissue and cellular gene expression. Recently, it has been shown that DG is produced in various subcellular compartments including the plasma membrane, internal membranes, cytoskeleton, and nucleus. However, it remains unclear how DG is regulated at distinct subcellular sites. To address this point, we have used an epitope-tag expression system in cultured cells and investigated the subcellular localization of DGK isozymes under the same experimental conditions. We show here that DGK isozymes are targeted differentially to unique subcellular sites in transfected COS7 cells, including the cytoplasm, actin stress fibers, Golgi complex, endoplasmic reticulum, and nucleus. It is also shown that among the isozymes overexpression of DGKbeta causes fragmentation of actin stress fibers while a kinase-dead mutant of DGKbeta abolishes its colocalization with actin stress fibers. These data strongly suggest that each isozyme may be responsible for the metabolism of DG that is produced upon stimulation at a different and specific subcellular site and that DGKbeta activity might have effects on the reorganization of actin stress fibers in transfected COS7 cells.     

A chimeric elongation factor containing the putative guanine nucleotide binding domain of archaeal EF-1 alpha and the M and C domains of eubacterial EF-Tu.

A recombinant chimeric elongation factor containing the region of EF-1 alpha from Sulfolobus solfataricus harboring the site for GDP and GTP binding and GTP hydrolysis (SsG) and domains M and C of Escherichia coli EF-Tu (EcMC) was studied. SsG-EcMC did not sustain poly(Phe) synthesis in either S. solfataricus or E. coli assay system. This was probably due to the inability of the chimera to interact with aa-tRNA. The three-dimensional modeling of SsG-EcMC indicated only small structural differences compared to the Thermus aquaticus EF-Tu in the ternary complex with aa-tRNA and GppNHp, which did not account for the observed inability to interact with aa-tRNA. The addition of the nucleotide exchange factor SsEF-1 beta was not required for poly(Phe) synthesis since the chimera was already able to exchange [(3)H]GDP for GTP at very high rate even at 0 degrees C. Compared to that of SsEF-1 alpha, the affinity of the chimera for guanine nucleotides was increased and the k(cat) of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 degrees C lower than that displayed by SsG and SsEF-1alpha, respectively, but 30 degrees C higher than that of EcEF-Tu. This pattern remained almost the same if the melting curves of the proteins being investigated were considered instead. The chimeric elongation factor was more thermophilic than SsG and SsEF-1 alpha up to 70 degrees C; at higher temperatures, inactivation occurred.     

The DNA topoisomerase I binding protein topors as a novel cellular target for SUMO-1 modification: characterization of domains necessary for subcellular localization and sumolation

Over the past years, modification by covalent attachment of SUMO (small ubiquitin-like modifier) has been demonstrated for of a number of cellular and viral proteins. While increasing evidence suggests a role for SUMO modification in the regulation of protein-protein interactions and/or subcellular localization, most SUMO targets are still at large. In this report we show that Topors, a Topoisomerase I and p53 interacting protein of hitherto unknown function, presents a novel cellular target for SUMO-1 modification. In a yeast two-hybrid system, Topors interacted with both SUMO-1 and the SUMO-1 conjugating enzyme UBC9. Multiple SUMO-1 modified forms of Topors could be detected after cotransfection of exogenous SUMO-1 and Topors induced the colocalization of a YFP tagged SUMO-1 protein in a speckled pattern in the nucleus. A subset of these Topors' nuclear speckles were closely associated with the PML nuclear bodies (POD, ND10). A central domain comprising Topors residues 437 to 574 was sufficient for both sumolation and localization to nuclear speckles. One SUMO-1 acceptor site at lysine residue 560 could be identified within this region. However, sumolation-deficient Topors mutants showed that sumolation obviously is not required for localization to nuclear speckles.     

Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide

We have isolated cis-acting mutations in the gene encoding the yeast vacuolar protein carboxypeptidase Y (CPY) that result in missorting and aberrant secretion of up to 95% of newly synthesized CPY. The CPY polypeptides synthesized by these mutants use the late secretory pathway to exit the cell, since the late-acting sec1 mutation prevents their secretion. The mutant versions of CPY are secreted as the proCPY zymogen and are enzymatically activatable in vivo and in vitro. All the mutations, including small deletions and an amino acid substitution, map to the amino-terminal propeptide region and define a discrete yeast vacuolar localization domain whose integrity is required for efficient sorting of the CPY zymogen. Thus, the N-terminal propeptide of CPY carries out at least three functions: it mediates translocation across the endoplasmic reticulum, renders the enzyme inactive during transit, and targets the molecule to the vacuole.     

Protein domains,catalytic activity,and subcellular distribution of neuropathy target esterase in Mammalian cells

Neuropathy target esterase (NTE), the human homologue of a protein required for brain development in Drosophila, has a predicted amino-terminal transmembrane helix (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. Here we describe the expression, in COS cells, of green fluorescent protein-tagged constructs of NTE and mutant proteins lacking the TM or the R- or C-domains. Esterase assays and Western blots of particulate and soluble fractions indicated that neither the TM nor R-domain is essential for NTE catalytic activity but that this activity requires membrane association to which the TM, R-, and C-domains all contribute. Experiments involving proteinase treatment revealed that most of the NTE molecule is exposed on the cytoplasmic face of membranes. In cells expressing a moderate level of NTE and all cells expressing DeltaC-NTE, fluorescence was distributed in an endoplasmic reticulum (ER)-like pattern. Cells expressing high levels of NTE showed aberrant distribution of ER marker proteins and accumulation of NTE on the cytoplasmic surface of ER-derived tubuloreticular aggregates. Deformation of the ER was also seen in cells expressing DeltaR-NTE or enzymatically inactive S966A-NTE but not DeltaTM-NTE. The data suggest that NTE is anchored in the ER via its TM, that its R- and C-domains also interact with the cytoplasmic face of the ER, and that overexpression of NTE causes ER aggregation via intermolecular association of its C-domains.     

Transaldolase genes from the cyanobacteria Anabaena variabilis and Synechocystis sp. PCC 6803: comparison with other eubacterial and eukaryotic homologues

We have sequenced and analysed the transaldolase (tal) genes from two cyanobacteria, Anabaena variabilis (ATCC 29413) and Synechocystis sp. PCC 6803, which are filamentous heterocyst-forming and unicellular organisms, respectively. The deduced amino acid sequences of the two cyanobacterial tal genes are 78% identical and are highly homologous to both eubacterial and eukaryotic transaldolases (Escherichia coli, two yeasts, and man) with values ranging from 54 to 60% amino acid identity. In contrast, the transaldolase homologous sequences from the cyanobacterium Nostoc sp. ATCC 29133, from Mycobacterium leprae, and the partial sequence from the higher plant Arabidopsis thaliana have a much lower degree of homology with each other and relative to the sequences mentioned above. These data indicate three different types of transaldolases.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

A single eubacterial origin of eukaryotic pyruvate: ferredoxin oxidoreductase genes: implications for the evolution of anaerobic eukaryotes.

The iron sulfur protein pyruvate: ferredoxin oxidoreductase (PFO) is central to energy metabolism in amitochondriate eukaryotes, including those with hydrogenosomes. Thus, revealing the evolutionary history of PFO is critical to understanding the origin(s) of eukaryote anaerobic energy metabolism. We determined a complete PFO sequence for Spironucleus barkhanus, a large fragment of a PFO sequence from Clostridium pasteurianum, and a fragment of a new PFO from Giardia lamblia. Phylogenetic analyses of eubacterial and eukaryotic PFO genes suggest a complex history for PFO, including possible gene duplications and horizontal transfers among eubacteria. Our analyses favor a common origin for eukaryotic cytosolic and hydrogenosomal PFOs from a single eubacterial source, rather than from separate horizontal transfers as previously suggested. However, with the present sampling of genes and species, we were unable to infer a specific eubacterial sister group for eukaryotic PFO. Thus, we find no direct support for the published hypothesis that the donor of eukaryote PFO was the common alpha-proteobacterial ancestor of mitochondria and hydrogenosomes. We also report that several fungi and protists encode proteins with PFO domains that are likely monophyletic with PFOs from anaerobic protists. In Saccharomyces cerevisiae, PFO domains combine with fragments of other redox proteins to form fusion proteins which participate in methionine biosynthesis. Our results are consistent with the view that PFO, an enzyme previously considered to be specific to energy metabolism in amitochondriate protists, was present in the common ancestor of contemporary eukaryotes and was retained, wholly or in part, during the evolution of oxygen-dependent and mitochondrion-bearing lineages.     

Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: Limited interdomain misfolding in a eukaryotic translation system

It has been proposed that eukaryotic translation systems have a greater capacity for cotranslational folding of domains than prokaryotic translation systems, which reduces interdomain misfolding in multidomain proteins and, therefore, leads to tolerance for random recombination of domains. However, there has been a controversy as to whether prokaryotic and eukaryotic translation systems differ in the capacity for cotranslational domain folding. Here, to examine whether these systems differ in the tolerance for the random domain recombination, we systematically combined six proteins, out of which four are soluble and two are insoluble when produced in an Escherichia coli and a wheat germ cell-free protein synthesis systems, to construct a fusion protein library. Forty out of 60 two-domain proteins and 114 out of 120 three-domain proteins were more soluble when produced in the wheat system than in the E. coli system. Statistical analyses of the solubilities and the activities indicated that, in the wheat system but not in the E. coli system, the two soluble domains comprised mainly of beta-sheets tend to avoid interdomain misfolding and to fold properly even at the neighbor of the misfolded domains. These results demonstrate that a eukaryotic system permits the concomitance of a wider variety of domains within a single polypeptide chain than a prokaryotic system, which is probably due to the difference in the capacity for cotranslational folding. This difference is likely to be related to the postulated difference in the tolerance for random recombination of domains.