scholarly journals Protein localization to the nucleolus: a search for targeting domains in nucleolin

1993 ◽  
Vol 105 (3) ◽  
pp. 799-806 ◽  
Author(s):  
M.S. Schmidt-Zachmann ◽  
E.A. Nigg
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Protein Localization ◽  
Site Directed Mutagenesis ◽  
Rdna Transcription ◽  
Binding Motifs ◽  
Mutant Proteins ◽  
C Terminus ◽  
Localization Signal ◽  
Functional Nuclear Localization Signal

Nucleolin, a major nucleolar phosphoprotein, is presumed to function in rDNA transcription, rRNA packaging and ribosome assembly. Its primary sequence was highly conserved during evolution and suggests a multi-domain structure. To identify structural elements required for nuclear uptake and nucleolar accumulation of nucleolin, we used site-directed mutagenesis to introduce point- and deletion-mutations into a chicken nucleolin cDNA. Following transient expression in mammalian cells, the intracellular distribution of the corresponding wild-type and mutant proteins was determined by indirect immunofluorescence microscopy. We found that nucleolin contains a functional nuclear localization signal (KRKKEMANKSAPEAKKKK) that conforms exactly to the consensus proposed recently for a bipartite signal (Robbins, J., Dilworth, S.M., Laskey, R.A. and Dingwall, C. (1991) Cell 64, 615–623). Concerning nucleolar localization, we found that the N-terminal 250 amino acids of nucleolin are dispensible, but deletion of either the centrally located RNA-binding motifs (the RNP domain) or the glycine/arginine-rich C terminus (the GR domain) resulted in an exclusively nucleoplasmic distribution. Although both of these latter domains were required for correct subcellular localization of nucleolin, they were not sufficient to target non-nucleolar proteins to the nucleolus. From these results we conclude that nucleolin does not contain a single, linear nucleolar targeting signal. Instead, we propose that the protein uses a bipartite NLS to enter the nucleus and then accumulates within the nucleolus by virtue of binding to other nucleolar components (probably rRNA) via its RNP and GR domains.

scholarly journals The RNA Binding Protein Nuclear Factor 90 Functions as Both a Positive and Negative Regulator of Gene Expression in Mammalian Cells

2002 ◽  
Vol 22 (1) ◽  
pp. 343-356 ◽  
Author(s):  
Trevor W. Reichman ◽  
Luis C. Muñiz ◽  
Michael B. Mathews
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Interleukin 2 ◽  
Negative Regulator ◽  
Nuclear Factor ◽  
C Terminus ◽  
Inhibit Gene Expression ◽  
Localization Signal ◽  
Major Late Promoter

ABSTRACT Nuclear factor 90 (NF90) was originally isolated in a complex that binds to the antigen recognition response element (ARRE-2) present in the interleukin-2 promoter. To characterize the transcriptional properties of NF90 in mammalian cells, we examined its ability to modulate promoter function in cellular transfection assays. NF90-Gal4 fusion proteins inhibited transcription from the adenovirus major late promoter in a fashion that was dependent on Gal4 targeting. Conversely, NF90 activated the cytomegalovirus immediate-early promoter, to which it was not targeted. These effects required distinct but overlapping domains in the C terminus of NF90, which contains a functional nuclear localization signal and two double-stranded-RNA binding motifs. NF90 is present in cellular complexes together with the NF45 protein. Transfection assays showed that NF45 binds NF90 strongly and stimulates its ability to activate but not to inhibit gene expression. This report characterizes NF90 as both a positive and negative regulator of gene expression, depending on the promoter context, and suggests a role for NF45 as a regulator of NF90.

scholarly journals MOLECULAR DETERMINANTS OF THE ENDOCYTIC PROTEIN EPSIN CONTROLLING ITS LOCALIZATION AND FUNCTION IN CANCER CELL MIGRATION AND INVASION

2020 ◽  
Author(s):  
Kayalvizhi Madhivanan ◽  
Lingyan Cao ◽  
Chris J. Staiger ◽  
R. Claudio Aguilar
Keyword(s):  
Cell Migration ◽  
Mammalian Cells ◽  
Protein Localization ◽  
Adaptor Proteins ◽  
Lipid Binding ◽  
Migration And Invasion ◽  
Binding Motifs ◽  
Enth Domain ◽  
C Terminus ◽  
And Function

ABSTRACTEpsins are endocytic adaptor proteins with signaling and endocytic functions. The three mammalian epsin paralogs are made of an Epsin N-Terminal Homology (ENTH) domain and an unstructured C-terminal region. The highly conserved ENTH domain plays a role in signaling by blocking RhoGAP activity and is required for cell migration in mammalian cells. However, our lab has previously shown that only epsin full length overexpression can enhance cell migration, but the ENTH domain alone cannot. Among the three Epsin paralogs, epsin 3 followed by epsin 2 were able to substantially enhance cell migration. This study is the first one to systematically and comprehensibly address the contribution of different motifs within the epsin C-terminus to enhance protein localization and cell migration. We show that is not the lipid-binding ENTH domain, but the C-terminus of epsin the one playing a major role in epsin association with sites of endocytosis. Further, we dissected the contribution of individual C-terminal endocytic (clathrin-, AP2-, Ubiquitin- and EH domain-binding) motifs for epsin localization. We found that while all motifs show a degree of synergism, the clathrin-binding motifs are the most important for epsin localization. Our study also showed that, these motifs (particularly the clathrin binding site) play an important role in sustaining endocytic site dynamics and cell migration.

scholarly journals Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

2005 ◽  
Vol 393 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Catherine Martel ◽  
Paolo Macchi ◽  
Luc Furic ◽  
Michael A. Kiebler ◽  
Luc Desgroseillers
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Binding Activity ◽  
Nuclear Trafficking ◽  
Binding Domains ◽  
Bipartite Nuclear Localization Signal ◽  
Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

scholarly journals Sla1, a Schizosaccharomyces pombe Homolog of the Human La Protein, Induces Ectopic Meiosis when Its C Terminus Is Truncated

2003 ◽  
Vol 2 (6) ◽  
pp. 1274-1287 ◽  
Author(s):  
Kaori Tanabe ◽  
Noriko Ito ◽  
Tomomi Wakuri ◽  
Fumiyo Ozoe ◽  
Makoto Umeda ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Small Region ◽  
Full Length ◽  
Rna Recognition Motifs ◽  
La Protein ◽  
C Terminus ◽  
Localization Signal ◽  
Recognition Motifs

ABSTRACT Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1ΔC) induced ectopic sporulation in the ras1Δ strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1ΔC is found throughout the cell, suggesting the cytoplasmic localization of Sla1ΔC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1ΔC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1ΔC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1ΔC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1ΔC but not full-length Sla1. Thus, Sla1ΔC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.

scholarly journals Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates

2021 ◽  
Vol 8 ◽  
Author(s):  
Sabine Panzer ◽  
Chong Zhang ◽  
Tilen Konte ◽  
Celine Bräuer ◽  
Anne Diemar ◽  
...  
Keyword(s):  
Proton Pump ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Aureobasidium Pullulans ◽  
Green Light ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Proton Pumps ◽  
C Terminus ◽  
Pump Activity

Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

scholarly journals A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.

1994 ◽  
Vol 14 (12) ◽  
pp. 8399-8407 ◽  
Author(s):  
J Flach ◽  
M Bossie ◽  
J Vogel ◽  
A Corbett ◽  
T Jinks ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Binding Protein ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Binding Motifs ◽  
C Terminus ◽  
N Terminus ◽  
Heterogeneous Nuclear Ribonucleoproteins

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.

scholarly journals Identification and Functional Characterization of a Novel Nuclear Localization Signal Present in the Yeast Nab2 Poly(A)+ RNA Binding Protein

1998 ◽  
Vol 18 (3) ◽  
pp. 1449-1458 ◽  
Author(s):  
Ray Truant ◽  
Robert A. Fridell ◽  
R. Edward Benson ◽  
Hal Bogerd ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Yeast Cells ◽  
Vitro Assay ◽  
Localization Signal

ABSTRACT The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin α/importin α, which acts as the NLS receptor, and karyopherin β1/importin β, which binds karyopherin α and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin β1, termed karyopherin β2 or transportin, and does not require a karyopherin α-like adapter protein. A yeast homolog of karyopherin β2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin β1, but not the Kap104p homolog karyopherin β2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin α. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

scholarly journals Unexpected Role for a Serine/Threonine-Rich Domain in the Candida albicans Iff Protein Family

2011 ◽  
Vol 10 (10) ◽  
pp. 1317-1330 ◽  
Author(s):  
Anita Boisramé ◽  
Amandine Cornu ◽  
Grégory Da Costa ◽  
Mathias L. Richard
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Protein Localization ◽  
Protein Family ◽  
Content Type ◽  
Rich Domain ◽  
C Terminus ◽  
The Family ◽  
Localization Signal ◽  
First Time

ABSTRACTGlycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins inCandida albicansbecause of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag on all members of the family. The data obtained using the complete proteins showed for Iff3 (or -9), Iff5, Iff6, and Iff8 a covalent linkage to the β-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOH-labile bonds. However, since some proteins of the Iff family were undetectable, we designed chimeric constructions using the last 60 amino acids of these proteins to test the localization signal. These constructions showed a β-1,6-glucan linkage for Iff1/Rbr3, Iff2/Hyr3, Iff4 and Iff7/Hyr4 C-terminal–Iff5 fusion proteins, and a membrane localization for the Iff10/Flo9 C terminus-Iff5 fusion protein. Immunofluorescence analyses coupled to these cell fraction data confirmed the importance of the length of the central serine/threonine-rich region for cell surface exposure. Further analysis of the Iff2/Hyr3 linkage to the cell surface showed for the first time that a serine/threonine central region of a GPI-anchored protein may be responsible for the disulfide and the NaOH bonds to the glucan and glycoproteins network and may also override the signal of the proximal ω site region.

scholarly journals A cysteine near the C-terminus of UCH-L1 is dispensable for catalytic activity but is required to promote AKT phosphorylation, eIF4F assembly, and malignant B-cell survival

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Sajjad Hussain ◽  
Tibor Bedekovics ◽  
Asma Ali ◽  
Omar Zaid ◽  
Danielle G. May ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Survival ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Supporting Cell ◽  
Site Directed Mutagenesis ◽  
Akt Phosphorylation ◽  
Molecular Features ◽  
C Terminus ◽  
Metabolic Activities

AbstractThe enzyme UCH-L1 is a neuro-endocrine and germinal center B-cell marker that contributes to the development and aggressive behavior of mature B-cell malignancies. While mutations in this enzyme have been associated with Parkinson’s disease, relatively little is known about the molecular features associated with the biochemical activities of UCH-L1. Here we use a survival-based complementation assay and site-directed mutagenesis and identify a novel role for the C-terminus of UCH-L1 in supporting cell survival. The C220 residue is required for UCH-L1 to promote the assembly of mTOR complex 2 and phosphorylation of the pro-survival kinase AKT. While this residue was previously described as a potential farnesylation site, destruction of the putative CAAX motif by adding a C-terminal epitope tag did not interfere with cell survival, indicating an alternate mechanism. We used proximity-based proteomics comparing the proteomes of wild-type and C220S UCH-L1 and identified a selective loss of association with RNA-binding proteins including components of the translation initiation machinery. As a consequence, the C220S mutant did not promote the assembly of the eIF4F complex. These data identify a novel role for the C-terminus of UCH-L1 in supporting pro-survival and metabolic activities in malignant B-cells. This finding may lead to the development of therapeutics with selective activity towards malignancy that potentially avoid neuronal toxicities.

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