Regulated nuclear import of the Drosophila rel protein dorsal: structure-function analysis.

The formation of a gradient of nuclear Dorsal protein in the early Drosophila embryo is the last step in a maternally encoded dorsal-ventral signal transduction pathway. This gradient is formed in response to a ventral signal, which leads to the dissociation of cytoplasmic Dorsal from the I kappa B homolog Cactus. Free Dorsal is then targeted to the nucleus. Dorsal is a Rel-family transcription factor. Signal-dependent nuclear localization characterizes the regulation of Rel proteins. In order to identify regions of Dorsal that are essential for its homodimerization, nuclear targeting, and interaction with Cactus, we have performed an in vivo structure-function analysis. Our results show that all these functions are carried out by regions within the conserved Rel-homology region of Dorsal. The C-terminal divergent half of Dorsal is dispensable for its selective nuclear import. A basic stretch of 6 amino acids at the C terminus of the Rel-homology region is necessary for nuclear localization. This nuclear localization signal is not required for Cactus binding. Removal of the N-terminal 40 amino acids abolished the nuclear import of Dorsal, uncovering a potentially novel function for this highly conserved region.

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Structure/Function Analysis of the Vaccinia Virus F18 Phosphoprotein, an Abundant Core Component Required for Virion Maturation and Infectivity

Journal of Virology ◽

10.1128/jvi.00399-10 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6846-6860 ◽

Cited By ~ 15

Author(s):

Nadi T. Wickramasekera ◽

Paula Traktman

Keyword(s):

Amino Acids ◽

Structure Function ◽

Protein Interactions ◽

Function Analysis ◽

Aromatic Amino Acids ◽

Protein Protein Interactions ◽

Core Component ◽

Virion Morphogenesis

ABSTRACT Poxvirus virions, whose outer membrane surrounds two lateral bodies and a core, contain at least 70 different proteins. The F18 phosphoprotein is one of the most abundant core components and is essential for the assembly of mature virions. We report here the results of a structure/function analysis in which the role of conserved cysteine residues, clusters of charged amino acids and clusters of hydrophobic/aromatic amino acids have been assessed. Taking advantage of a recombinant virus in which F18 expression is IPTG (isopropyl-β-d-thiogalactopyranoside) dependent, we developed a transient complementation assay to evaluate the ability of mutant alleles of F18 to support virion morphogenesis and/or to restore the production of infectious virus. We have also examined protein-protein interactions, comparing the ability of mutant and WT F18 proteins to interact with WT F18 and to interact with the viral A30 protein, another essential core component. We show that F18 associates with an A30-containing multiprotein complex in vivo in a manner that depends upon clusters of hydrophobic/aromatic residues in the N′ terminus of the F18 protein but that it is not required for the assembly of this complex. Finally, we confirmed that two PSSP motifs within F18 are the sites of phosphorylation by cellular proline-directed kinases in vitro and in vivo. Mutation of both of these phosphorylation sites has no apparent impact on virion morphogenesis but leads to the assembly of virions with significantly reduced infectivity.

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The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains.

The Journal of Cell Biology ◽

10.1083/jcb.131.5.1261 ◽

1995 ◽

Vol 131 (5) ◽

pp. 1261-1273 ◽

Cited By ~ 40

Author(s):

K Oegema ◽

W G Whitfield ◽

B Alberts

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Nuclear Localization ◽

Fusion Proteins ◽

Drosophila Embryo ◽

Dependent Manner ◽

Fluorescence Confocal Microscopy ◽

Early Drosophila Embryo ◽

A Cell ◽

Cycle Dependent

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19-amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.

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Structure-function analysis of TAF130: identification and characterization of a high-affinity TATA-binding protein interaction domain in the N terminus of yeast TAF(II)130.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3081 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3081-3093 ◽

Cited By ~ 55

Author(s):

Y Bai ◽

G M Perez ◽

J M Beechem ◽

P A Weil

Keyword(s):

Amino Acids ◽

Cell Growth ◽

Structure Function ◽

Function Analysis ◽

Binding Protein ◽

Single Copy ◽

Tata Binding Protein ◽

Interaction Domain ◽

Support Cell

We report structure-function analyses of TAF130, the single-copy essential yeast gene encoding the 130,000-Mr yeast TATA-binding protein (TBP)-associated factor TAF(II)130 (yTAF(II)130). A systematic family of TAF130 mutants was generated, and these mutant TAF130 alleles were introduced into yeast in both single and multiple copies to test for their ability to complement a taf130delta null allele and support cell growth. All mutant proteins were stably expressed in vivo. The complementation tests indicated that a large portion (amino acids 208 to 303 as well as amino acids 367 to 1037) of yTAF(II)130 is required to support cell growth. Direct protein blotting and coimmunoprecipitation analyses showed that two N-terminal deletions which remove portions of yTAF(II)130 amino acids 2 to 115 dramatically decrease the ability of these mutant yTAF(II)130 proteins to bind TBP. Cells bearing either of these two TAF130 mutant alleles also exhibit a slow-growth phenotype. Consistent with these observations, overexpression of TBP can correct this growth deficiency as well as increase the amount of TBP interacting with yTAF(II)130 in vivo. Our results provide the first combined genetic and biochemical evidence that yTAF(II)130 binds to yeast TBP in vivo through yTAF(II)130 N-terminal sequences and that this binding is physiologically significant. By using fluorescence anisotropy spectroscopic binding measurements, the affinity of the interaction of TBP for the N-terminal TBP-binding domain of yTAF(II)130 was measured, and the Kd was found to be about 1 nM. Moreover, we found that the N-terminal domain of yTAF(II)130 actively dissociated TBP from TATA box-containing DNA.

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Signal- and importin-dependent nuclear targeting of the kidney anion exchanger 1-binding protein kanadaptin

Biochemical Journal ◽

10.1042/bj3610287 ◽

2002 ◽

Vol 361 (2) ◽

pp. 287-296 ◽

Cited By ~ 6

Author(s):

Stefan HÜBNER ◽

David A. JANS ◽

Chong-Yun XIAO ◽

Anna P. JOHN ◽

Detlev DRENCKHAHN

Keyword(s):

Amino Acids ◽

Fusion Protein ◽

Nuclear Localization ◽

Nuclear Import ◽

Anion Exchanger ◽

Cell Types ◽

Nuclear Targeting ◽

Anion Exchanger 1 ◽

Cultured Cell ◽

Importin Α

Kanadaptin (kidney anion exchanger adaptoror protein) has recently been identified as a protein with binding activity to the cytoplasmic domain of the kidney Na+-independent Cl−/HCO−3 anion exchanger 1 (kAE1). Since it is widely expressed in tissues devoid of kAE1, however, kanadaptin is likely to have additional cellular roles. This is supported by its multidomain structure, and possession of three clusters of basic amino acids exhibiting similarity to known nuclear localization sequences (NLSs). In the present study, we use immunofluorescence and subcellular fractionation approaches to demonstrate that kanadaptin is localized within the nuclei of various epithelial and non-epithelial cultured cell types. The role of the different NLSs is examined in transfection studies using plasmids encoding full-length kanadaptin with or without green fluorescent protein (GFP) as a fusion tag, as well as truncation derivatives thereof. Strong nuclear localization of fusion proteins containing amino acids 140–230 of kanadaptin, which include the sequence AVSRKRKA193 (NLS1) was observed. Substitution of Arg191 with a threonine residue resulted in a cytoplasmic location of the expressed protein, while NLS1 proved sufficient to target an otherwise cytoplasmically localized β-galactosidase—GFP fusion protein to the nucleus. Using a direct binding assay we show that a fusion protein containing kanadaptin amino acids 1–231 (but not the Thr191 substituted derivative) is recognized with nM affinity by the conventional NLS-binding importin α/β heterodimer. Nuclear import studies on microinjected and permeabilized rat hepatoma cells demonstrated functionality of the NLS in nuclear targeting, with inhibition by antibodies demonstrating the requirement of both importin α and β for nuclear import of kanadaptin. That kanadaptin possesses a functional importin-α/β-recognized NLS explains the nuclear localization of kanadaptin in various cultured cell types, and opens up the possibility that kanadaptin may have a signalling role in the nucleus.

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FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles

Biochemical Journal ◽

10.1042/bj20042040 ◽

2005 ◽

Vol 388 (2) ◽

pp. 509-514 ◽

Cited By ~ 10

Author(s):

Kathryn L. SUNN ◽

John A. EISMAN ◽

Edith M. GARDINER ◽

David A. JANS

Keyword(s):

Vitamin D ◽

Vitamin D Receptor ◽

Nuclear Import ◽

Protein Import ◽

Nucleocytoplasmic Transport ◽

Nuclear Speckles ◽

Nuclear Targeting ◽

Nuclear Retention ◽

First Time

Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B1) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)-containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.

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Quantitative Analysis of Gene Function in the Drosophila Embryo

Genetics ◽

10.1093/genetics/154.1.273 ◽

2000 ◽

Vol 154 (1) ◽

pp. 273-284

Author(s):

William D Tracey ◽

Xiangqun Ning ◽

Martin Klingler ◽

Sunita G Kramer ◽

J Peter Gergen

Keyword(s):

Gene Function ◽

Model Systems ◽

Drosophila Embryo ◽

Direct Role ◽

Developmental Context ◽

Maternal Mrna ◽

Early Drosophila Embryo ◽

Segment Polarity ◽

Pair Rule

Abstract The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.

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Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo

The Journal of Cell Biology ◽

10.1083/jcb.200608141 ◽

2006 ◽

Vol 175 (4) ◽

pp. 579-593 ◽

Cited By ~ 100

Author(s):

Benjamin L. Timney ◽

Jaclyn Tetenbaum-Novatt ◽

Diana S. Agate ◽

Rosemary Williams ◽

Wenzhu Zhang ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Pore Complex ◽

Nuclear Import ◽

Yeast Cells ◽

Limiting Factor ◽

Nuclear Pore ◽

Nuclear Localization Signals ◽

The Poor ◽

Cytoplasmic Proteins

Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump–leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm.

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The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro

Biochemistry and Cell Biology ◽

10.1139/o96-039 ◽

1996 ◽

Vol 74 (3) ◽

pp. 363-372 ◽

Cited By ~ 9

Author(s):

Werner Barth ◽

Ursula Stochaj

Keyword(s):

Nuclear Localization ◽

Specific Binding ◽

T Antigen ◽

Nuclear Localization Sequence ◽

Nuclear Pore ◽

Sv40 T Antigen ◽

Nuclear Targeting ◽

Nuclear Localization Sequences

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 Gal4. 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 × 107 M−1 and 5 × 107 M−1. An association constant of 5 × 107 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.Key words: Nsp1, nuclear pore complex, nucleoporin, nuclear localization sequence, protein targeting, yeast.

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Nuclear Import of Cdk/Cyclin Complexes: Identification of Distinct Mechanisms for Import of Cdk2/Cyclin E and Cdc2/Cyclin B1

The Journal of Cell Biology ◽

10.1083/jcb.144.2.213 ◽

1999 ◽

Vol 144 (2) ◽

pp. 213-224 ◽

Cited By ~ 130

Author(s):

Jonathan D. Moore ◽

Jing Yang ◽

Ray Truant ◽

Sally Kornbluth

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Cyclin E ◽

Cyclin B1 ◽

Transport Processes ◽

Nuclear Localization Sequence ◽

Nuclear Targeting ◽

Importin Α

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence–containing protein, binding to the α adaptor subunit of the importin-α/β heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-β that is distinct from that used to bind importin-α.

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Essential Role of Nuclear Localization for Yeast Ulp2 SUMO Protease Function

Molecular Biology of the Cell ◽

10.1091/mbc.e08-10-1090 ◽

2009 ◽

Vol 20 (8) ◽

pp. 2196-2206 ◽

Cited By ~ 28

Author(s):

Mary B. Kroetz ◽

Dan Su ◽

Mark Hochstrasser

Keyword(s):

Nuclear Localization ◽

Nuclear Protein ◽

Catalytic Domain ◽

Yeast Growth ◽

Nuclear Targeting ◽

Sumo Protease ◽

Sumo Proteases ◽

Localization Signal

The SUMO protein is covalently attached to many different substrates throughout the cell. This modification is rapidly reversed by SUMO proteases. The Saccharomyces cerevisiae SUMO protease Ulp2 is a nuclear protein required for chromosome stability and cell cycle restart after checkpoint arrest. Ulp2 is related to the human SENP6 protease, also a nuclear protein. All members of the Ulp2/SENP6 family of SUMO proteases have large but poorly conserved N-terminal domains (NTDs) adjacent to the catalytic domain. Ulp2 also has a long C-terminal domain (CTD). We show that CTD deletion has modest effects on yeast growth, but poly-SUMO conjugates accumulate. In contrast, the NTD is essential for Ulp2 function and is required for nuclear targeting. Two short, widely separated sequences within the NTD confer nuclear localization. Efficient Ulp2 import into the nucleus requires the β-importin Kap95, which functions on classical nuclear-localization signal (NLS)-bearing substrates. Remarkably, replacement of the entire >400-residue NTD by a heterologous NLS results in near-normal Ulp2 function. These data demonstrate that nuclear localization of Ulp2 is crucial in vivo, yet only small segments of the NTD provide the key functional elements, explaining the minimal sequence conservation of the NTDs in the Ulp2/SENP6 family of enzymes.

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