005.Efficiency of nuclear import of the chromatin-remodelling factor SRY is critical for sex determination

2005 ◽  
Vol 17 (9) ◽  
pp. 64
Author(s):  
D. A. Jans ◽  
G. Kaur ◽  
I. K. H. Poon ◽  
A. Delluc-Clavieries ◽  
K. M. Wagstaff
Keyword(s):  
Sex Determination ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Sex Reversal ◽  
Nuclear Accumulation ◽  
Missense Mutations ◽  
Nuclear Localization Signals ◽  
Testicular Cells ◽  
Xy Sex Reversal

15% of cases of human XY sex reversal are due to mutations in SRY (sex determining region on the Y chromosome), many of which map to one of SRY’s two independently acting nuclear localization signals (NLSs) flanking its DNA binding domain. The C-terminal NLS (C-NLS) targets SRY to the nucleus through a ‘conventional’ pathway dependent on the nuclear import receptor importin-β (Imp-β). No importin has been shown to bind the N-terminal NLS (N-NLS), but it is known to interact with the Ca2+-binding protein calmodulin (CaM). We examined seven distinct missense mutations in the SRY NLSs from XY sex-reversed human females for effects on nuclear import and ability to interact with CaM/Imp-β1. All mutations were found to result in reduced nuclear localization in transfected testicular cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce SRY nuclear accumulation, indicating a dependence of SRY nuclear import on CaM. Intriguingly, N-NLS mutants were resistant to CDZ’s effects, implying a loss of interaction with CaM; this was confirmed directly by in vitro binding experiments using recombinantly expressed protein. Either impaired CaM or Imp-β1 binding can thus be the basis of sex-reversal in human patients. Our results implicate a CaM-dependent nuclear import pathway for SRY mediated by the N-NLS that, together with the C-NLS, is required to achieve threshold levels of SRY in the nucleus for male sex determination.

scholarly journals Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

2005 ◽  
Vol 19 (7) ◽  
pp. 1884-1892 ◽  
Author(s):  
Helena Sim ◽  
Kieran Rimmer ◽  
Sabine Kelly ◽  
Louisa M. Ludbrook ◽  
Andrew H. A. Clayton ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Nuclear Import ◽  
High Mobility ◽  
Sex Reversal ◽  
High Mobility Group ◽  
Missense Mutations ◽  
Nuclear Localization Signals ◽  
Xy Sex Reversal

Abstract The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-β binding to the C-terminal NLS (c-NLS), whereas in others, importin-β recognition is normal, suggesting the existence of an importin-β-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

scholarly journals Inhibition of SRY-Calmodulin Complex Formation Induces Ectopic Expression of Ovarian Cell Markers in Developing XY Gonads

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2883-2893 ◽  
Author(s):  
Helena Sim ◽  
Anthony Argentaro ◽  
Daniel P. Czech ◽  
Stefan Bagheri-Fam ◽  
Andrew H. Sinclair ◽  
...  
Keyword(s):  
Sex Determination ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Transcriptional Activity ◽  
Ectopic Expression ◽  
Disorders Of Sex Development ◽  
Gonadal Development ◽  
Nuclear Accumulation ◽  
Transfected Cells ◽  
Nuclear Localization Signals

The transcription factor sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, because mutations in SRY cause disorders of sex development in XY individuals. During gonadal development, Sry in pre-Sertoli cells activates Sox9 gene transcription, committing the fate of the bipotential gonad to become a testis rather than an ovary. The high-mobility group domain of human SRY contains two independent nuclear localization signals, one bound by calmodulin (CaM) and the other by importin-β. Although XY females carry SRY mutations in these nuclear localization signals that affect SRY nuclear import in transfected cells, it is not known whether these transport mechanisms are essential for gonadal development and sex determination. Here, we show that mouse Sry protein binds CaM and that a CaM antagonist reduces CaM binding, nuclear accumulation, and transcriptional activity of Sry in transfected cells. CaM antagonist treatment of cultured, sexually indifferent XY mouse fetal gonads led to reduced expression of the Sry target gene Sox9, defects in testicular cord formation, and ectopic expression of the ovarian markers Rspondin1 and forkhead box L2. These results indicate the importance of CaM for SRY nuclear import, transcriptional activity, testis differentiation, and sex determination.

scholarly journals The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals

1999 ◽  
Vol 19 (2) ◽  
pp. 1210-1217 ◽  
Author(s):  
Ray Truant ◽  
Bryan R. Cullen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signals ◽  
Immunodeficiency Virus ◽  
Importin Α

ABSTRACT Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin α (Imp α) NLS receptor. Imp α is in turn bound by importin β (Imp β), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp α and instead bind directly to Imp β. Using in vitro nuclear import assays, we demonstrate that Imp α is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp β proved both sufficient and necessary, in that other β-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp β for Imp α, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp β is also similar to Imp α binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp β and that therefore function independently of Imp α.

scholarly journals The CUL1 C-Terminal Sequence and ROC1 Are Required for Efficient Nuclear Accumulation, NEDD8 Modification, and Ubiquitin Ligase Activity of CUL1

2000 ◽  
Vol 20 (21) ◽  
pp. 8185-8197 ◽  
Author(s):  
Manabu Furukawa ◽  
Yanping Zhang ◽  
Joseph McCarville ◽  
Tomohiko Ohta ◽  
Yue Xiong
Keyword(s):  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Nuclear Import ◽  
Ring Finger ◽  
Nuclear Accumulation ◽  
Protein Families ◽  
Terminal Sequence ◽  
Ubiquitin Ligase Activity

ABSTRACT Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IκBα ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity—ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.

scholarly journals The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal.

1995 ◽  
Vol 130 (2) ◽  
pp. 255-263 ◽  
Author(s):  
T Tagawa ◽  
T Kuroki ◽  
P K Vogt ◽  
K Chida
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Kinase Inhibitors ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Localization Signal ◽  
Cycle Dependent

Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.

scholarly journals Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin α

2005 ◽  
Vol 86 (6) ◽  
pp. 1815-1826 ◽  
Author(s):  
O. Guerra-Peraza ◽  
D. Kirk ◽  
V. Seltzer ◽  
K. Veluthambi ◽  
A. C. Schmit ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Fluorescent Protein ◽  
Yellow Mosaic Virus ◽  
Rice Tungro Bacilliform Virus ◽  
Coat Proteins ◽  
Nuclear Localization Signals ◽  
Mungbean Yellow Mosaic Virus

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant pararetro- and geminiviruses. In both these virus types, the multifunctional coat protein (CP) is thought to be involved in this process. Here, a green fluorescent protein tagging approach was used to demonstrate nuclear import of the CPs of Rice tungro bacilliform virus (RTBV) and Mungbean yellow mosaic virus - Vigna (MYMV) in Nicotiana plumbaginifolia protoplasts. In both cases, at least two nuclear localization signals (NLSs) were identified and characterized. The NLSs of RTBV CP are located within both N- and C-terminal regions (residues 479KRPK/497KRK and 744KRK/758RRK), and those of MYMV CP within the N-terminal part (residues 3KR and 41KRRR). The MYMV and RTBV CP NLSs resemble classic mono- and bipartite NLSs, respectively. However, the N-terminal MYMV CP NLS and both RTBV CP NLSs show peculiarities in the number and position of basic residues. In vitro pull-down assays revealed interaction of RTBV and MYMV CPs with the nuclear import factor importin α, suggesting that both CPs are imported into the nucleus via an importin α-dependent pathway. The possibility that this pathway could serve for docking of virions to the nucleus is discussed.

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish

1997 ◽  
Vol 75 (5) ◽  
pp. 633-640 ◽  
Author(s):  
Philippe Collas ◽  
Peter Aleström
Keyword(s):  
Nuclear Localization ◽  
Plasmid Dna ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Transport ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
Expression Frequency ◽  
Cytoplasmic Injection

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

scholarly journals Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions

2006 ◽  
Vol 87 (9) ◽  
pp. 2709-2720 ◽  
Author(s):  
Gideon Kass ◽  
Gabriel Arad ◽  
Joseph Rosenbluh ◽  
Yedidya Gafni ◽  
Adolf Graessmann ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Synthetic Peptides ◽  
Mammalian Cells ◽  
Specific Binding ◽  
Nuclear Localization Signals ◽  
Direct Demonstration ◽  
Bipartite Genome ◽  
Monopartite Genome

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two ∼2.6 kb DNAs or a monopartite genome of ∼3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3–20 of TYLCV CP (CP-NLS) or aa 84–106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA–NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin α, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin α.

scholarly journals Sequence Elements in Both Subunits of the DNA Fragmentation Factor Are Essential for Its Nuclear Transport

2007 ◽  
Vol 282 (49) ◽  
pp. 35821-35830 ◽  
Author(s):  
Sonja Neimanis ◽  
Werner Albig ◽  
Detlef Doenecke ◽  
Joerg Kahle
Keyword(s):  
Dna Fragmentation ◽  
Nuclear Import ◽  
Dna Cleavage ◽  
Nuclear Accumulation ◽  
Proliferating Cells ◽  
Nuclear Localization Signals ◽  
Importin Α ◽  
The Individual

DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin α/β-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin α/β-heterodimer. However, importin α/β binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin α/β more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin α/β with the DFF complex are presented.

scholarly journals Calmodulin-dependent nuclear import of HMG-box family nuclear factors: importance of the role of SRY in sex reversal

2010 ◽  
Vol 430 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Gurpreet Kaur ◽  
Aurelie Delluc-Clavieres ◽  
Ivan K. H. Poon ◽  
Jade K. Forwood ◽  
Dominic J. Glover ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Calcium Binding ◽  
Nuclear Translocation ◽  
Sex Reversal ◽  
Nuclear Accumulation ◽  
Common Mechanism ◽  
Wild Type ◽  
Hmg Box ◽  
First Time

The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located ‘β-NLS’ that mediates nuclear transport through Impβ1 (importin β1) and the N-terminally located ‘CaM-NLS’ which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)–SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors.

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