Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins

Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl− serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

Compositional differences within and between eukaryotic genomes

Eukaryotic genome similarity relationships are inferred using sequence information derived from large aggregates of genomic sequences. Comparisons within and between species sample sequences are based on the profile of dinucleotide relative abundance values (The profile is ρ*XY = f*XY/f*Xf*Y for all XY, where f*X denotes the frequency of the nucleotide X and f*XY denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complement). Previous studies with respect to prokaryotes and this study document that profiles of different DNA sequence samples (sample size ≥50 kb) from the same organism are generally much more similar to each other than they are to profiles from other organisms, and that closely related organisms generally have more similar profiles than do distantly related organisms. On this basis we refer to the collection {ρ*XY} as the genome signature. This paper identifies ρ*XY extremes and compares genome signature differences for a diverse range of eukaryotic species. Interpretations on the mechanisms maintaining these profile differences center on genome-wide replication, repair, DNA structures, and context-dependent mutational biases. It is also observed that mitochondrial genome signature differences between species parallel the corresponding nuclear genome signature differences despite large differences between corresponding mitochondrial and nuclear signatures. The genome signature differences also have implications for contrasts between rodents and other mammals, and between monocot and dicot plants, as well as providing evidence for similarities among fungi and the diversity of protists.

The α-helical FXXΦΦ motif in p53: TAF interaction and discrimination by MDM2

Transcriptional activation domains share little sequence homology and generally lack folded structures in the absence of their targets, aspects that have rendered activation domains difficult to characterize. Here, a combination of biochemical and nuclear magnetic resonance experiments demonstrates that the activation domain of the tumor suppressor p53 has an FXXΦΦ motif (F, Phe; X, any amino acids; Φ, hydrophobic residues) that folds into an α-helix upon binding to one of its targets, hTAFII31 (a human TFIID TATA box-binding protein-associated factor). MDM2, the cellular attenuator of p53, discriminates the FXXΦΦ motif of p53 from those of NF-κB p65 and VP16 and specifically inhibits p53 activity. Our studies support the notion that the FXXΦΦ sequence is a general α-helical recognition motif for hTAFII31 and provide insights into the mechanistic basis for regulation of p53 function.

Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver

Cholesterol feeding reduces the mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway and the low density lipoprotein receptor in livers of hamsters. Here we show that cholesterol feeding also reduces the levels of the nuclear NH2-terminal domains of sterol regulatory element binding proteins (SREBPs), which activate transcription of sterol-regulated genes. We show that livers of hamsters, like those of mice and humans, predominantly produce SREBP-2 and the 1c isoform of SREBP-1. Both are produced as membrane-bound precursors that must be proteolyzed to release the transcriptionally active NH2-terminal domains. Diets containing 0.1% to 1.0% cholesterol decreased the amount of nuclear SREBP-1c without affecting the amount of the membrane precursor or its mRNA, suggesting that cholesterol inhibits the proteolytic processing of SREBP-1 in liver as it does in cultured cells. Cholesterol also appeared to reduce the proteolytic processing of SREBP-2. In addition, at high levels of dietary cholesterol the mRNA encoding SREBP-2 declined and the amount of the precursor also fell, suggesting that cholesterol accumulation also may inhibit transcription of the SREBP-2 gene. The high-cholesterol diets reduced the amount of low density lipoprotein receptor mRNA by 30% and produced a more profound 70–90% reduction in mRNAs encoding 3-hydroxy-3-methylglutaryl CoA synthase and reductase. Treatment with lovastatin and Colestipol, which increases hepatic demands for cholesterol, increased the amount of SREBP-2 mRNA as well as the precursor and nuclear forms of the protein. This treatment caused a reciprocal decline in SREBP-1c mRNA and protein. Considered together, these data suggest that SREBPs play important roles in controlling transcription of sterol-regulated genes in liver, as they do in cultured cells.

Interplay of Signal Mediators of Decapentaplegic (Dpp): Molecular Characterization of Mothers against dpp, Medea, and Daughters against dpp

Decapentaplegic (Dpp) plays an essential role in Drosophila development, and analyses of the Dpp signaling pathway have contributed greatly to understanding of the actions of the TGF-β superfamily. Intracellular signaling of the TGF-β superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. Here we report the identification of a common mediator Smad in Drosophila, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Smad4 (Medea) upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp. Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. We also show that Mad exists as a monomer in the absence of Tkv stimulation. Tkv induces homo-oligomerization of Mad, and Dad inhibits this step. Finally, we propose a model for Dpp signaling by Drosophila Smad proteins.

Characterization of ATM Expression, Localization, and Associated DNA-dependent Protein Kinase Activity

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA–dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.

A Polymer Model for the Structural Organization of Chromatin Loops and Minibands in Interphase Chromosomes

A quantitative model of interphase chromosome higher-order structure is presented based on the isochore model of the genome and results obtained in the field of copolymer research. G1 chromosomes are approximated in the model as multiblock copolymers of the 30-nm chromatin fiber, which alternately contain two types of 0.5- to 1-Mbp blocks (R and G minibands) differing in GC content and DNA-bound proteins. A G1 chromosome forms a single-chain string of loop clusters (micelles), with each loop ∼1–2 Mbp in size. The number of ∼20 loops per micelle was estimated from the dependence of geometrical versus genomic distances between two points on a G1 chromosome. The greater degree of chromatin extension in R versus G minibands and a difference in the replication time for these minibands (early S phase for R versus late S phase for G) are explained in this model as a result of the location of R minibands at micelle cores and G minibands at loop apices. The estimated number of micelles per nucleus is close to the observed number of replication clusters at the onset of S phase. A relationship between chromosomal and nuclear sizes for several types of higher eukaryotic cells (insects, plants, and mammals) is well described through the micelle structure of interphase chromosomes. For yeast cells, this relationship is described by a linear coil configuration of chromosomes.

Phosphoinositide Signaling Pathways in Nuclei Are Associated with Nuclear Speckles Containing Pre-mRNA Processing Factors

Phosphoinositide signal transduction pathways in nuclei use enzymes that are indistinguishable from their cytosolic analogues. We demonstrate that distinct phosphatidylinositol phosphate kinases (PIPKs), the type I and type II isoforms, are concentrated in nuclei of mammalian cells. The cytosolic and nuclear PIPKs display comparable activities toward the substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate. Indirect immunofluorescence revealed that these kinases were associated with distinct subnuclear domains, identified as “nuclear speckles,” which also contained pre-mRNA processing factors. A pool of nuclear phosphatidylinositol bisphosphate (PIP2), the product of these kinases, was also detected at these same sites by monoclonal antibody staining. The localization of PIPKs and PIP2 to speckles is dynamic in that both PIPKs and PIP2 reorganize along with other speckle components upon inhibition of mRNA transcription. Because PIPKs have roles in the production of most phosphatidylinositol second messengers, these findings demonstrate that phosphatidylinositol signaling pathways are localized at nuclear speckles. Surprisingly, the PIPKs and PIP2 are not associated with invaginations of the nuclear envelope or any nuclear membrane structure. The putative absence of membranes at these sites suggests novel mechanisms for the generation of phosphoinositides within these structures.

Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

We have characterized HsCdc6, a human protein homologous to the budding yeast Cdc6p that is essential for DNA replication. We show that, unlike Cdc6p, the levels of HsCdc6 protein remain constant throughout the cell cycle in human cells. However, phosphorylation of HsCdc6 is regulated during the cell cycle. HsCdc6 is an excellent substrate for Cdk2 in vitro and is phosphorylated in vivo at three sites (Ser-54, Ser-74, and Ser-106) that are phosphorylated by Cdk2 in vitro, strongly suggesting that HsCdc6 is an in vivo Cdk substrate. HsCdc6 is nuclear in G1, but translocates to the cytoplasm at the start of S phase via Crm1-dependent export. An HsCdc6A1A2A3 mutant, which mimics unphosphorylated HsCdc6, is exclusively nuclear, and its expression inhibits initiation of DNA replication. An HsCdc6E1E2E3 mutant, which mimics phosphorylated HsCdc6, is exclusively cytoplasmic and is not associated with the chromatin/nuclear matrix fraction. Based on these results, we propose that phosphorylation of HsCdc6 by Cdks regulates DNA replication of at least two steps: first, by promoting initiation of DNA replication and, second, through nuclear exclusion preventing DNA rereplication.

Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

The functional role of the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in epidermal growth factor (EGF)-induced expression of 12(S)-lipoxygenase gene in human epidermoid carcinoma A431 cells was studied. Coimmunoprecipitation experiments indicated that EGF stimulated interaction between c-Jun and Sp1 in a time-dependent manner. Overexpression of Ha-ras and c-Jun also enhanced the amount of c-Jun binding to Sp1. In addition, the c-Jun dominant negative mutant TAM-67 not only inhibited the coimmunoprecipitated c-Jun binding to Sp1 in a dose-dependent manner in cells overexpressing c-Jun but also reduced promoter activity of the 12(S)-lipoxygenase gene induced by c-Jun overexpression. Treatment of cells with EGF increased the interaction between the Sp1 oligonucleotide and nuclear c-Jun/Sp1 in a time-dependent manner. Furthermore, EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12(S)lipoxygenase promoter only when coexpressed with GAL4-c-Jun () fusion proteins. These results indicate that the direct interaction between c-Jun and Sp1 induced by EGF cooperatively activated expression of the 12(S)-lipoxygenase gene, and that Sp1 may serve at least in part as a carrier bringing c-Jun to the promoter, thus transactivating the transcriptional activity of 12(S)-lipoxygenase gene.

Independent and combined analyses of sequences from all three genomic compartments converge on the root of flowering plant phylogeny

Plant phylogenetic estimates are most likely to be reliable when congruent evidence is obtained independently from the mitochondrial, plastid, and nuclear genomes with all methods of analysis. Here, results are presented from separate and combined genomic analyses of new and previously published data, including six and nine genes (8,911 bp and 12,010 bp, respectively) for different subsets of taxa that suggest Amborella + Nymphaeales (water lilies) are the first-branching angiosperm lineage. Before and after tree-independent noise reduction, most individual genomic compartments and methods of analysis estimated the Amborella + Nymphaeales basal topology with high support. Previous phylogenetic estimates placing Amborella alone as the first extant angiosperm branch may have been misled because of a series of specific problems with paralogy, suboptimal outgroups, long-branch taxa, and method dependence. Ancestral character state reconstructions differ between the two topologies and affect inferences about the features of early angiosperms.

Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis

Apoptosis is a highly regulated form of cell death, characterized by distinctive features such as cellular shrinkage and nuclear condensation. We demonstrate here that proteolytic activation of hPAK65, a p21-activated kinase, induces morphological changes and elicits apoptosis. hPAK65 is cleaved both in vitro and in vivo by caspases at a single site between the N-terminal regulatory p21-binding domain and the C-terminal kinase domain. The C-terminal cleavage product becomes activated, with a kinetic profile that parallels caspase activation during apoptosis. This C-terminal hPAK65 fragment also activates the c-Jun N-terminal kinase pathway in vivo. Microinjection or transfection of this truncated hPAK65 causes striking alterations in cellular and nuclear morphology, which subsequently promotes apoptosis in both CHO and Hela cells. Conversely, apoptosis is delayed in cells expressing a dominant-negative form of hPAK65. These findings provide a direct evidence that the activated form of hPAK65 generated by caspase cleavage is a proapoptotic effector that mediates morphological and biochemical changes seen in apoptosis.

Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus

Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “in vivo” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.

Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes

The influenza A virus pandemic of 1918–1919 resulted in an estimated 20–40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Evolutionary relationships among Rel domains indicate functional diversification by recombination

The recent sequencing of several complete genomes has made it possible to track the evolution of large gene families by their genomic structure. Following the large-scale association of exons encoding domains with well defined functions in invertebrates could be useful in predicting the function of complex multidomain proteins in mammals produced by accretion of domains. With this objective, we have determined the genomic structure of the 14 genes in invertebrates and vertebrates that contain rel domains. The sequence encoding the rel domain is defined by intronic boundaries and has been recombined with at least three structurally and functionally distinct genomic sequences to generate coding sequences for: (i) the rel/Dorsal/NFκB proteins that are retained in the cytoplasm by IkB-like proteins; (ii) the NFATc proteins that sense calcium signals and undergo cytoplasmic-to-nuclear translocation in response to dephosphorylation by calcineurin; and (iii) the TonEBP tonicity-responsive proteins. Remarkably, a single exon in each NFATc family member encodes the entire Ca2+/calcineurin sensing region, including nuclear import/export, calcineurin-binding, and substrate regions. The Rel/Dorsal proteins and the TonEBP proteins are present in Drosophila but not Caenorhabditis elegans. On the other hand, the calcium-responsive NFATc proteins are present only in vertebrates, suggesting that the NFATc family is dedicated to functions specific to vertebrates such as a recombinational immune response, cardiovascular development, and vertebrate-specific aspects of the development and function of the nervous system.