Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nuclear import factor p10 was cloned from Saccharomyces cerevisiae and found to be essential. The protein p10 can bind directly to several peptide repeat-containing nucleoporins. It also binds to the guanosine triphosphatase (GTPase) Ran in its guanosine diphosphate (GDP)-bound form and to karyopherin beta. Assembly of the karyopherin heterodimer on immobilized nucleoporin yielded cooperative binding of p10 and Ran-GDP. Addition of GTP to this pentameric complex led to dissociation of karyopherin (chi, presumably via in situ formation of Ran-GTP from Ran-GDP. Thus, p10 appears to coordinate the Ran-dependent association and dissociation reactions underlying nuclear import.
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PMID:Role of the nuclear transport factor p10 in nuclear import. 860 May 22

Facilitated transport of proteins into the nucleus requires nuclear localization sequences (NLSs) be present in the protein destined for the nucleus. The specific binding of NLSs by components of the nuclear transport apparatus is essential for these targeting reactions. We now report that the yeast nucleoporin Nsp1 binds specifically nuclear localization sequences in vitro. This nucleoporin recognizes several NLSs that are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Ga14. Nsp1 is organized into three domains, and we have located NLS binding sites to the N-terminal portion and the middle repetitive region of the protein. For the interaction between the NLS of SV40 T-antigen and Nsp1, we obtained association constants of 1.2 x 10(7) M-1 and 5 x 10(7) M-1. An association constant of 5 x 10(7) M-1 was determined for NLS binding to the repetitive domain of Nsp1. We analyzed binding of Nsp1 and its domains to a mutant version of the NLS derived from SV40 T-antigen, which poorly functions for nuclear targeting in vivo. The affinity for the mutant signal was about two orders of magnitude lower than for the wild-type NLS.
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PMID:The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro. 888 42

In this study, we report different protocols used to obtain highly enriched and well-characterized protein fractions that could be used to determine the subcellular localization of proteins. Different protein fractions (total, cytosolic, total membrane, sarcolemmal, and nuclear) were isolated from mouse heart by a combination of either polytron homogenization or liquid nitrogen pulverization followed by density gradient centrifugation. Triton X-100 was used in specific fractions to help in the solubilization of proteins obtained with fractionation protocols. Following the isolation, enzymatic assays and Western blot analysis were used to evaluate the enrichment and/or cross-contamination of these protein fractions. Glucose-6-phosphate dehydrogenase, Na+/K+-ATPase, mitochondrial Ca2+-ATPase, sarco-endoplasmic reticulum Ca2+-ATPase, glucose-regulated protein, and nucleoporin P62 were used as specific markers for the cytosol, sarcolemma, mitochondria, sarco-endoplasmic reticulum, endoplasmic reticulum, and nucleus, respectively. The results show that we obtained enriched protein fractions with little to no cross-contamination. These purification protocols allow us to obtain different protein fractions that could be used in a wide variety of studies.
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PMID:Isolation and characterization of subcellular protein fractions from mouse heart. 1612 24

Proteins can enter the nucleus through various receptor-mediated import pathways. One class of import cargos carries a classical nuclear localization signal (cNLS) containing a short cluster of basic residues. This pathway involves importin alpha (Impalpha), which possesses the cNLS binding site, and importin beta (Impbeta), which translocates the import complex through the nuclear pore complex. The defining criteria for a cNLS protein from Saccharomyces cerevisiae are an in vivo import defect in Impalpha and Impbeta mutants, direct binding to purified Impalpha, and stimulation of this binding by Impbeta. We show for the first time that endogenous S. cerevisiae proteins Prp20, Cdc6, Swi5, Cdc45, and Clb2 fulfill all of these criteria identifying them as authentic yeast cNLS cargos. Furthermore, we found that the targeting signal of Prp20 is a bipartite cNLS and that of Cdc6 is a monopartite cNLS. Basic residues present within these motifs are of different significance for the interaction with Impalpha. We determined the binding constants for import complexes containing the five cNLS proteins by surface plasmon resonance spectrometry. The dissociation constants for cNLS/alpha/beta complexes differ considerably, ranging from 1 nM for Cdc6 to 112 nM for Swi5, suggesting that the nuclear import kinetics is determined by the strength of cNLS/Impalpha binding. Impbeta enhances the affinity of Impalpha for cNLSs approximately 100-fold. This stimulation of cNLS binding to Impalpha results from a faster association in the presence of Impbeta, whereas the dissociation rate is unaffected by Impbeta. This implies that, after entry into the nucleus, the release of Impbeta by the Ran guanosine triphosphatase (Ran GTPase) from the import complex is not sufficient to dissociate the cNLS/Impalpha subcomplex. Our observation that the nucleoporin Nup2, which had been previously shown to release the cNLS from Impalpha in vitro, is required for efficient import of all the genuine cNLS cargos supports a general role of Nup2 in import termination.
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PMID:Classical NLS proteins from Saccharomyces cerevisiae. 1848 66

The DEAD-box protein DBP5 is essential for mRNA export in both yeast and humans. It binds RNA and is concentrated and locally activated at the cytoplasmic side of the nuclear pore complex. We have determined the crystal structures of human DBP5 bound to RNA and AMPPNP, and bound to the cytoplasmic nucleoporin NUP214. The structures reveal that binding of DBP5 to nucleic acid and to NUP214 is mutually exclusive. Using in vitro assays, we demonstrate that NUP214 decreases both the RNA binding and ATPase activities of DBP5. The interactions are mediated by conserved residues, implying a conserved recognition mechanism. These results suggest a framework for the consecutive steps leading to the release of mRNA at the final stages of nuclear export. More generally, they provide a paradigm for how binding of regulators can specifically inhibit DEAD-box proteins.
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PMID:The mRNA export protein DBP5 binds RNA and the cytoplasmic nucleoporin NUP214 in a mutually exclusive manner. 1921 46

Nuclear export of mRNA is a critical event in mRNA biogenesis. Passage of mature messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is facilitated by the Mex67/Mtr2 heterodimer. At the NPC cytoplasmic face, the DEAD-box RNA helicase Dbp5 remodels mRNPs by removing Mex67/Mtr2. This remodeling process prevents mRNPs from returning to the nucleus, thereby imposing unidirectionality on mRNA export. Biochemical studies show that Gle1 and inositol hexaphosphate (IP6) activate Dbp5's ATPase activity at the cytoplasmic face of NPC, therefore providing critical spatial regulation of mRNP remodeling during directional transport. Recent structural studies on Dbp5 in free form and in complex with its ligands (ADP, AMPPNP/RNA) as well as with cytoplasmic nucleoporin Nup214 reveal that the binding of ADP or AMPPNP/RNA induces large conformational changes of Dbp5, and RNA and NUP214 bind to Dbp5 in a mutually exclusive manner. These structural data combined with complementary functional analysis significantly advance our understanding on the mechanism governing mRNA export albeit some key issues remain elusive.
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PMID:Mechanistic insights into mRNA export through structures of Dbp5. 2002

Early embryogenesis in Arabidopsis (Arabidopsis thaliana) is distinguished by a predictable pattern of cell divisions and is a good system for investigating mechanisms of developmental pattern formation. Here, we identified a gene called LONO1 (LNO1) in Arabidopsis in which mutations can abolish the first asymmetrical cell division of the zygote, alter planes and number of cell divisions in early embryogenesis, and eventually arrest embryo development. LNO1 is highly expressed in anthers of flower buds, stigma papilla of open flowers, and embryo and endosperm during early embryogenesis, which is correlated with its functions in reproductive development. The homozygous lno1-1 seed is not viable. LNO1, a homolog of the nucleoporin NUP214 in human (Homo sapiens) and Nup159 in yeast (Saccharomyces cerevisiae), encodes a nucleoporin protein containing phenylalanine-glycine repeats in Arabidopsis. We demonstrate that LNO1 can functionally complement the defect in the yeast temperature-sensitive nucleoporin mutant nup159. We show that LNO1 specifically interacts with the Arabidopsis DEAD-box helicase/ATPase LOS4 in the yeast two-hybrid assay. Furthermore, mutations in AtGLE1, an Arabidopsis homolog of the yeast Gle1 involved in the same poly(A) mRNA export pathway as Nup159, also result in seed abortion. Our results suggest that LNO1 is a component of the nuclear pore complex required for mature mRNA export from the nucleus to the cytoplasm, which makes LNO1 essential for embryogenesis and seed viability in Arabidopsis.
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PMID:LONO1 encoding a nucleoporin is required for embryogenesis and seed viability in Arabidopsis. 2289 97

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.
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PMID:Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation. 2318 31

mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.
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PMID:P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5). 2673 Aug 86

Beyond their role at nuclear pore complexes, some nucleoporins function in the nucleoplasm. One such nucleoporin, Nup98, binds chromatin and regulates gene expression. To gain insight into how Nup98 contributes to this process, we focused on identifying novel binding partners and understanding the significance of these interactions. Here we report on the identification of the DExH/D-box helicase DHX9 as an intranuclear Nup98 binding partner. Various results, including in vitro assays, show that the FG/GLFG region of Nup98 binds to N- and C-terminal regions of DHX9 in an RNA facilitated manner. Importantly, binding of Nup98 stimulates the ATPase activity of DHX9, and a transcriptional reporter assay suggests Nup98 supports DHX9-stimulated transcription. Consistent with these observations, our analysis revealed that Nup98 and DHX9 bind interdependently to similar gene loci and their transcripts. Based on our results, we propose that Nup98 functions as a co-factor that regulates DHX9 and, potentially, other RNA helicases.
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PMID:Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9. 2822 Nov 34


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