3'-Phosphoadenosine 5'-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding

Activated sulfate in the form of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is needed for all sulfation reactions in eukaryotes with implications for the build-up of extracellular matrices, retroviral infection, protein modification, and steroid metabolism. In metazoans, PAPS is produced by bifunctional PAPS synthases (PAPSS). A major question in the field is why two human protein isoforms, PAPSS1 and -S2, are required that cannot complement for each other. We provide evidence that these two proteins differ markedly in their stability as observed by unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism spectroscopy. At 37 °C, the half-life for unfolding of PAPSS2 is in the range of minutes, whereas PAPSS1 remains structurally intact. In the presence of their natural ligand, the nucleotide adenosine 5'-phosphosulfate (APS), PAPS synthase proteins are stabilized. Invertebrates only possess one PAPS synthase enzyme that we classified as PAPSS2-type by sequence-based machine learning techniques. To test this prediction, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermal unfolding. With respect to thermal unfolding and the stabilization by APS, PPS-1 behaved like the unstable human PAPSS2 protein suggesting that the less stable protein is evolutionarily older. Finally, APS binding more than doubled the half-life for unfolding of PAPSS2 at physiological temperatures and effectively prevented its aggregation on a time scale of days. We propose that protein stability is a major contributing factor for PAPS availability that has not as yet been considered. Moreover, naturally occurring changes in APS concentrations may be sensed by changes in the conformation of PAPSS2.     

A heterodimer of human 3′-phospho-adenosine-5′-phosphosulphate (PAPS) synthases is a new sulphate activating complex

3′-Phospho-adenosine-5′-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5′-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.     

Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.

Sulfation is a major modification of many molecules in eukaryotes that is dependent on the enzymatic synthesis of an activated sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). While sulfate activation has long been assumed to occur in the cytosol, we show in this study that human PAPS synthetase 1 (PAPSS1), a bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme sufficient for PAPS synthesis, accumulates in the nucleus of mammalian cells. Nuclear targeting of the enzyme is mediated by its APS kinase domain and requires a catalytically dispensable 21 amino acid sequence at the amino terminus. Human PAPSS1 and Drosophila melanogaster PAPSS localize to the nucleus in yeast and relieve the methionine auxotrophy of ATP sulfurylase- or APS kinase-deficient strains, suggesting that PAPSS1 is fully functional in vivo when targeted to the nucleus. A second PAPS synthetase gene, designated PAPSS2, has recently been described, mutations of which are responsible for abnormal skeletal development in human spondyloepimetaphyseal dysplasia and murine brachymorphism. We found that PAPSS2, which localizes to the cytoplasm when ectopically expressed in mammalian cells, is relocated to the nucleus when coexpressed with PAPSS1. Taken together, these results indicate that a sulfation pathway might exist in the nucleus of eukaryotic cells. -Besset, S., Vincourt, J.-B., Amalric, F., Girard, J.-P. Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.     

Antagonistic control of flowering time by functionally specialized poly(A) polymerases in Arabidopsis thaliana

Polyadenylation is a critical 3′‐end processing step during maturation of pre‐mRNAs, and the length of the poly(A) tail affects mRNA stability, nuclear export and translation efficiency. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A) polymerase (PAPS) isoforms fulfilling specialized functions, as reflected by their different mutant phenotypes. While PAPS1 affects several processes, such as the immune response, organ growth and male gametophyte development, the roles of PAPS2 and PAPS4 are largely unknown. Here we demonstrate that PAPS2 and PAPS4 promote flowering in a partially redundant manner. The enzymes act antagonistically to PAPS1, which delays the transition to flowering. The opposite flowering‐time phenotypes in paps1 and paps2 paps4 mutants are at least partly due to decreased or increased FLC activity, respectively. In contrast to paps2 paps4 mutants, plants with increased PAPS4 activity flower earlier than the wild‐type, concomitant with reduced FLC expression. Double mutant analyses suggest that PAPS2 and PAPS4 act independently of the autonomous pathway components FCA, FY and CstF64. The direct polyadenylation targets of the three PAPS isoforms that mediate their effects on flowering time do not include FLC sense mRNA and remain to be identified. Thus, our results uncover a role for canonical PAPS isoforms in flowering‐time control, raising the possibility that modulating the balance of the isoform activities could be used to fine tune the transition to flowering.     

Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1

Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.     

Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine-rich nuclear export signals in Arabidopsis thaliana

Transport across the nuclear envelope is mediated by transport receptors from the Importin beta family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine-rich nuclear export signals (NESs). AtXPO1 shares 42-50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two-hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine-rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L176A, L179A, V181A). In BY-2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants.     

Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases

3′-phosphoadenosine 5′-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5′-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3′-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3′-OH requires Mg₂⁺ and purported to involve residues D₈₇GD₈₉N. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D₈₇ and D₈₉ did not hamper ATPS activity however abolished APSK activity severely. Thus, D₈₇GD₈₉N region is required for stabilization of Mg²⁺-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3′-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gamma³²P-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg²⁺ and APS of wild type and mutants supports the experimental results.     

The Host Cell Sulfonation Pathway Contributes to Retroviral Infection at a Step Coincident with Provirus Establishment

The early steps of retrovirus replication leading up to provirus establishment are highly dependent on cellular processes and represent a time when the virus is particularly vulnerable to antivirals and host defense mechanisms. However, the roles played by cellular factors are only partially understood. To identify cellular processes that participate in these critical steps, we employed a high volume screening of insertionally mutagenized somatic cells using a murine leukemia virus (MLV) vector. This approach identified a role for 3′-phosphoadenosine 5′-phosphosulfate synthase 1 (PAPSS1), one of two enzymes that synthesize PAPS, the high energy sulfate donor used in all sulfonation reactions catalyzed by cellular sulfotransferases. The role of the cellular sulfonation pathway was confirmed using chemical inhibitors of PAPS synthases and cellular sulfotransferases. The requirement for sulfonation was mapped to a stage during or shortly after MLV provirus establishment and influenced subsequent gene expression from the viral long terminal repeat (LTR) promoter. Infection of cells by an HIV vector was also shown to be highly dependent on the cellular sulfonation pathway. These studies have uncovered a heretofore unknown regulatory step of retroviral replication, have defined a new biological function for sulfonation in nuclear gene expression, and provide a potentially valuable new target for HIV/AIDS therapy.     

Histone deacetylase 4 possesses intrinsic nuclear import and export signals

Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.     

Subcellular localisation of human inositol 1,4,5-trisphosphate 3-kinase C: species-specific use of alternative export sites for nucleo-cytoplasmic shuttling indicates divergent roles of the catalytic and N-terminal domains

The three isoforms of human Ins(1,4,5)P3 3-kinase (IP3K) show remarkable differences in their intracellular targeting. Whereas predominant targeting to the cytoskeleton and endoplasmic reticulum has been shown for IP3K-A and IP3K-B, rat IP3K-C shuttles actively between the nucleus and cytoplasm. In the present study we examined the expression and intracellular localisation of endogenous IP3K-C in different mammalian cell lines using an isoform-specific antibody. In addition, human IP3K-C, showing remarkable differences to its rat homologue in the N-terminal targeting domain, was tagged with EGFP and used to examine active transport mechanisms into and out of the nucleus. We found both a nuclear import activity residing in its N-terminal domain and a nuclear export activity sensitive to treatment with leptomycin B. Different from the rat isoform, an exportin 1-dependent nuclear export site of the human enzyme resides outside the N-terminal targeting domain in the catalytic enzyme domain. A phylogenetic survey of vertebrate IP3K sequences indicates that in each of the three isoforms a nuclear export signal has evolved in the catalytic domain either de novo (IP3K-A) or as a substitute for an earlier evolved corresponding N-terminal signal (IP3K-B and IP3K-C). In higher vertebrates, and in particular in primates, re-export of nuclear IP3K activity may be guaranteed by the mechanism discovered.     

Molecular cloning of a novel human PAPS synthetase which is differentially expressed in metastatic and non-metastatic colon carcinoma cells.

Subtractive hybridisation was used to select for genes which are differentially expressed between a highly metastatic human colon carcinoma cell line, KM12SM, and the isogenetic non-metastatic cell line, KM12C. This led to the isolation of cDNA clones for a novel human adenosine 5'-phosphosulphate kinase/ATP sulphurylase (PAPS synthetase). Northern hybridisation revealed a single 4.2 kb mRNA species which showed an approximately 20-fold higher level of expression in the non-metastatic cell line than in the metastatic cell line. The overlapping cDNA clones together covered 3,774 bp including the entire coding region of 1,842 bp encoding a protein of 614 amino acids (calculated molecular mass of 69,496 Da). The protein contains consensus sequences for APS kinase and ATP sulphurylase, in its amino- and carboxy-terminal regions, respectively, as well as other sequences that are highly conserved amongst ATP sulphurylases and APS kinases. Interestingly, consensus sequences for GTPase activity were also identified, indicating that enzyme activity may be regulated by an intrinsic GTPase mechanism. Overall the new protein is 78% homologous with a previously described human PAPS synthetase (PAPSS1) indicating that we have identified the second member of a gene family which we have provisionally named PAPSS2. The gene locus for PAPSS2 was identified on chromosome 10 at 10q23.1-q23.2. This locus has synteny with the mouse brachymorphic gene recently identified as a PAPS synthetase (SK2). PAPSS2 appears to be the human homologue of this gene and thus PAPSS2 is likely to be important in human skeletogenesis.     

Molecular evidence for the nuclear localization of FADD

The Fas-associated death domain (FADD) adaptor protein FADD/Mort-1 is recruited by several members of the tumor necrosis factor receptor (TNFR) superfamily during cell death activated via death receptors. Since most studies have focused on the interaction of FADD with plasma membrane proteins, FADD's subcellular location is thought to be confined to the cytoplasm. In this report, we show for the first time that FADD is present in both the cytoplasm and the nucleus of cells, and that its nuclear localization relies on strong nuclear localization and nuclear export signals (NLS and NES, respectively) that reside in the death-effector domain (DED) of the protein. Specifically, we found that a conserved basic KRK35 sequence of the human protein is necessary for FADD's nuclear localization, since disruption of this motif leads to the confinement of FADD in the cytoplasm. Furthermore, we show that the leucine-rich motif LTELKFLCL28 in the DED is necessary for FADD's nuclear export. Functionally, mutation of the NES of FADD and its seclusion in the nucleus reduces the cell death-inducing efficacy of FADD reconstituted in FADD-deficient T cells.     

The nuclear export signal of splicing factor Uap56p interacts with nuclear pore-associated protein Rae1p for mRNA export in Schizosaccharomyces pombe

Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.     

Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import

The Cdc25 phosphatases play crucial roles in cell cycle progression by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Cdc25A is an important regulator of the G1/S transition but functions also in the mitotic phase of the human cell cycle. In this paper, we investigate the sub-cellular localisation of exogenously expressed Cdc25A. We show that YFP-Cdc25A is localised both in the nucleus and the cytoplasm of HeLa cells and untransformed fibroblasts. Cell fusion assays and fluorescence loss in photobleaching (FLIP) assays reveal that the localisation is dynamic and the protein shuttles between the nucleus and the cytoplasm. We demonstrate that nuclear export of Cdc25A is partly mediated by an N-terminal nuclear export sequence (NES), in a manner not sensitive to the Exportin 1-inhibitor leptomycin B. A nuclear localisation signal (NLS) is also characterised, mutation of which leads to cytoplasmic localisation of Cdc25A. Our results imply that the Cdc25A phosphatase may interact with substrates and regulators both in the nucleus and the cytoplasm.     

TAP binds to the constitutive transport element (CTE) through a novel RNA-binding motif that is sufficient to promote CTE-dependent RNA export from the nucleus

The constitutive transport element (CTE) of the simian type D retroviruses overcomes nuclear retention and allows nuclear export of unspliced viral RNAs by recruiting TAP, a host factor which is thought to be required for export of cellular mRNAs. In this report, we show that the first 372 amino acid residues of TAP, comprising a stretch of leucine-rich repeats, are both necessary and sufficient for binding to the CTE RNA and promoting its export to the cytoplasm. Moreover, like the full-length protein, this domain migrates to the cytoplasm upon nuclear co-injection with the CTE RNA. Together, these results indicate that the CTE-binding domain includes the signals for nuclear export. We also describe a derivative of TAP that bears a triple amino acid substitution within the CTE-binding domain and substantially reduces the export of mRNAs from the nucleus. This provides further evidence for a role for TAP in this process. Thus, the CTE-binding domain of TAP defines a novel RNA-binding motif which has dual functions, both recognizing the CTE RNA and interacting with other components of the nuclear transport machinery.