The subcellular localization of acetyl-CoA carboxylase 2

Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC 6.4.1.2), ACC1 (Mr = 265 kDa) and ACC2 (Mr = 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1–20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC–green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein. A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons. Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear. To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein. The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation. Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation. Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation. However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments. More dramatically, the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.

Signal recognition particle components in the nucleolus

The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain. The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72. SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing. Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized. It was therefore of interest to determine whether other SRP components localize to this intranuclear site. In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor. SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus. In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus. These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus. SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds. Our findings support the notion that the nucleolus is the site of assembly and/or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves.

Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the “A” fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.

Dynamic and quantitative Ca2+ measurements using improved cameleons

Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+-buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.

A deoxyribonucleotidase in mitochondria: Involvement in regulation of dNTP pools and possible link to genetic disease

Three cytosolic and one plasma membrane-bound 5′-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5′-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5′- and 2′(3′)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith–Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.

Inhibition of proteasomal degradation by the Gly-Ala repeat of Epstein–Barr virus is influenced by the length of the repeat and the strength of the degradation signal

The Gly-Ala repeat (GAr) of the Epstein–Barr virus nuclear antigen-1 is a transferable element that inhibits in cis ubiquitin/proteasome-dependent proteolysis. We have investigated this inhibitory activity by using green fluorescent protein-based reporters that have been targeted for proteolysis by N end rule or ubiquitin-fusion degradation signals, resulting in various degrees of destabilization. Degradation of the green fluorescent protein substrates was inhibited on insertion of a 25-aa GAr, but strongly destabilized reporters were protected only partially. Protection could be enhanced by increasing the length of the repeat. However, reporters containing the Ub-R and ubiquitin-fusion degradation signals were degraded even in the presence of a 239-aa GAr. In accordance, insertion of a powerful degradation signal relieved the blockade of proteasomal degradation in Epstein–Barr virus nuclear antigen-1. Our findings suggest that the turnover of natural substrates may be finely tuned by GAr-like sequences that counteract targeting signals for proteasomal destruction.

V(D)J recombination is not activated by demethylation of the kappa locus

V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation, Dnmt1, was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.

Retrovirus-mediated gene transfer of MLL-ELL transforms primary myeloid progenitors and causes acute myeloid leukemias in mice

The MLL-ELL fusion gene results from the translocation t(11;19)(q23;p13.1) that is associated with de novo and therapy-related acute myeloid leukemia. To study its transforming properties, we retrovirally transduced primary murine hematopoietic progenitors and assessed their growth properties both in vitro and in vivo. MLL-ELL increased the proliferation of myeloid colony-forming cells in methylcellulose cultures upon serial replating, whereas overexpression of ELL alone had no effect. We reconstituted lethally irradiated congenic mice with bone marrow progenitors transduced with MLL-ELL or the control MIE vector encoding the enhanced green fluorescent protein. When the peripheral blood of the mice was analyzed 11–13 weeks postreconstitution, we found that the engraftment of the MLL-ELL-transduced cells was superior to that of the MIE controls. At this time point, the contribution of the donor cells was normally distributed among the myeloid and nonmyeloid compartments. Although all of the MIE animals (n = 10) remained healthy for more than a year, all of the MLL-ELL mice (n = 20) succumbed to monoclonal or pauciclonal acute myeloid leukemias within 100–200 days. The leukemic cells were readily transplantable to secondary recipients and could be established as immortalized cell lines in liquid cultures. These studies demonstrate the enhancing effect of MLL-ELL on the proliferative potential of myeloid progenitors as well as its causal role in the genesis of acute myeloid leukemias.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.

Visualizing the dynamics of T cell activation: Intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes

We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.

Reconstitution of HIV-1 Rev nuclear export: Independent requirements for nuclear import and export

The Rev protein of HIV-1 actively shuttles between nucleus and cytoplasm and mediates the export of unspliced retroviral RNAs. The localization of shuttling proteins such as Rev is controlled by the relative rates of nuclear import and export. To study nuclear export in isolation, we generated cell lines expressing a green fluorescent protein-labeled chimeric protein consisting of HIV-1 Rev and a hormone-inducible nuclear localization sequence. Steroid removal switches off import thus allowing direct visualization of the Rev export pathway in living cells. After digitonin permeabilization of these cells, we found that a functional nuclear export sequence (NES), ATP, and fractionated cytosol were sufficient for nuclear export in vitro. Nuclear pore-specific lectins and leptomycin B were potent export inhibitors. Nuclear export was not inhibited by antagonists of calcium metabolism that block nuclear import. These data further suggest that nuclear pores do not functionally close when luminal calcium stores are depleted. The distinct requirements for nuclear import and export argue that these competing processes may be regulated independently. This system should have wide applicability for the analysis of nuclear import and export.

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl–tRNA synthetase in Arabidopsis thaliana

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl–tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl–tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNAMet genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl–tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.

Aldol sensors for the rapid generation of tunable fluorescence by antibody catalysis

The synthesis of novel fluorogenic retro-aldol substrates for aldolase antibody 38C2 is described. These substrates are efficiently and specifically processed by antibody aldolases but not by natural cellular enzymes. Together, the fluorogenic substrates and antibody aldolases provide reporter gene systems that are compatible with living cells. The broad scope of the antibody aldolase allows for the processing of a range of substrates that can be designed to allow fluorescence monitoring at a variety of wavelengths. We also have developed the following concept in fluorescent protein tags. β-Diketones bearing a fluorescent tag are bound covalently by the aldolase antibody and not other proteins. We anticipate that proteins fused with the antibody can be tagged specifically and covalently within living cells with fluorophores of virtually any color, thereby providing an alternative to green fluorescent protein fusions.