Bipolar localization of Bacillus subtilis topoisomerase IV, an enzyme required for chromosome segregation

In Bacillus subtilis, parE and parC were shown to be essential genes for the segregation of replicated chromosomes. Disruption of either one of these genes resulted in failure of the nucleoid to segregate. Purified ParE and ParC proteins reconstituted to form topoisomerase IV (topo IV), which was highly proficient for ATP-dependent superhelical DNA relaxation and decatenation of interlocked DNA networks. By immunofluorescence microscopy and by directly visualizing fluorescence by using green fluorescence protein fusions, we determined that ParC is localized at the poles of the bacteria in rapidly growing cultures. The bipolar localization of ParC required functional ParE, suggesting that topo IV activity is required for the localization. ParE was found to be distributed uniformly throughout the cell. On the other hand, fluorescence microscopy showed that the GyrA and GyrB subunits of gyrase were associated with the nucleoid. Our results provide a physiologic distinction between DNA gyrase and topo IV. The subcellular localization of topo IV provides physical evidence that it may be part of the bacterial segregation machinery.

Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism

Mammalian capping enzymes are bifunctional proteins with both RNA 5′-triphosphatase and guanylyltransferase activities. The N-terminal 237-aa triphosphatase domain contains (I/V)HCXXGXXR(S/T)G, a sequence corresponding to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Analysis of point mutants of mouse RNA 5′-triphosphatase identified the motif Cys and Arg residues and an upstream Asp as required for activity. Like PTPs, this enzyme was inhibited by iodoacetate and VO43− and independent of Mg2+, providing additional evidence for phosphate removal from RNA 5′ ends by a PTP-like mechanism. The full-length, 597-aa mouse capping enzyme and the C-terminal guanylyltransferase fragment (residues 211–597), unlike the triphosphatase domain, bound poly (U) and were nuclear in transfected cells. RNA binding was increased by GTP, and a guanylylation-defective, active-site mutant was not affected. Ala substitution at positions required for the formation of the enzyme-GMP capping intermediate (R315, R530, K533, or N537) also eliminated poly (U) binding, while proteins with conservative substitutions at these sites retained binding but not guanylyltransferase activity. These results demonstrate that the guanylyltransferase domain of mammalian capping enzyme specifies nuclear localization and RNA binding. Association of capping enzyme with nascent transcripts may act in synergy with RNA polymerase II binding to ensure 5′ cap formation.

A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

Localization and activity of lysyl oxidase within nuclei of fibrogenic cells

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.

Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II

Superoxide anion (O2−) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O2− is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O2− production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O2−-generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22phox, gp91phox, p47phox, and p67phox localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O2− production. Furthermore, immunodepletion of p67phox from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67phox. Further study into the regulation of this adventitial source of O2− is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.

Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme β-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls.  Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

A Ubiquitin-Conjugating Enzyme Is Essential for Developmental Transitions in Dictyostelium

We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for ∼10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.

Localization of α1,3-fucosyltransferase VI in Weibel–Palade bodies of human endothelial cells

Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked α1→3 to N-acetylglucosamine. A family of α1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription–PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with β4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel–Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel–Palade bodies suggests a function independent of Golgi-associated glycosylation.

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.

Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites such as Toxoplasma gondii contain a primitive plastid, the apicoplast, whose genome consists of a 35-kb circular DNA related to the plastid DNA of plants. Plants synthesize fatty acids in their plastids. The first committed step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC). This enzyme is encoded in the nucleus, synthesized in the cytosol, and transported into the plastid. In the present work, two genes encoding ACC from T. gondii were cloned and the gene structure was determined. Both ORFs encode multidomain proteins, each with an N-terminal extension, compared with the cytosolic ACCs from plants. The N-terminal extension of one isozyme, ACC1, was shown to target green fluorescent protein to the apicoplast of T. gondii. In addition, the apicoplast contains a biotinylated protein, consistent with the assertion that ACC1 is localized there. The second ACC in T. gondii appears to be cytosolic. T. gondii mitochondria also contain a biotinylated protein, probably pyruvate carboxylase. These results confirm the essential nature of the apicoplast and explain the inhibition of parasite growth in cultured cells by herbicides targeting ACC.

Intracellular Localization of Phospholipase D1 in Mammalian Cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.

Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification

Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH2 terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation.

Localization of GAR transformylase in Escherichia coli and mammalian cells

Enzymes of the de novo purine biosynthetic pathway may form a multienzyme complex to facilitate substrate flux through the ten serial steps constituting the pathway. One likely strategy for complex formation is the use of a structural scaffold such as the cytoskeletal network or subcellular membrane of the cell to mediate protein–protein interactions. To ascertain whether this strategy pertains to the de novo purine enzymes, the localization pattern of the third purine enzyme, glycinamide ribonucleotide transformylase (GAR Tfase) was monitored in live Escherichia coli and mammalian cells. Genes encoding human as well as E. coli GAR Tfase fused with green fluorescent protein (GFP) were introduced into their respective cells with regulated expression of proteins and localization patterns monitored by using confocal fluorescence microscopy. In both instances images showed proteins to be diffused throughout the cytoplasm. Thus, GAR Tfase is not localized to an existing cellular architecture, so this device is probably not used to concentrate the members of the pathway. However, discrete clusters of the pathway may still exist throughout the cytoplasm.

Purification and Characterization of NADP+-Linked Isocitrate Dehydrogenase from Scots Pine1 : Evidence for Different Physiological Roles of the Enzyme in Primary Development

NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic efficiency (105 m−1 s−1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.