scholarly journals Nucleolar Trafficking of Nucleostemin Family Proteins: Common versus Protein-Specific Mechanisms

2007 ◽  
Vol 27 (24) ◽  
pp. 8670-8682 ◽  
Author(s):  
Lingjun Meng ◽  
Qubo Zhu ◽  
Robert Y. L. Tsai
Keyword(s):  
Coiled Coil ◽  
Dependent Manner ◽  
Guanine Nucleotide Binding Protein ◽  
Binding Domains ◽  
Gtp Binding ◽  
Coiled Coil Domain ◽  
Versus Protein ◽  
Localization Signal ◽  
Guanine Nucleotide Binding ◽  
Retention Signal

ABSTRACT The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins.

scholarly journals Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow-induced Ca2+ signaling

2007 ◽  
Vol 292 (3) ◽  
pp. F930-F945 ◽  
Author(s):  
Chang Xu ◽  
Sandro Rossetti ◽  
Lianwei Jiang ◽  
Peter C. Harris ◽  
Ursa Brown-Glaberman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nk Cells ◽  
Coiled Coil ◽  
Human Kidney ◽  
Coiled Coil Domain ◽  
Normal Human Kidney ◽  
Cyst Cells ◽  
Localization Signal ◽  
Normal Human ◽  
Autosomal Dominant Polycystic Kidney

Autosomal dominant polycystic kidney disease (ADPKD) gene products polycystin-1 (PC1) and polycystin-2 (PC2) colocalize in the apical monocilia of renal epithelial cells. Mouse and human renal cells without PC1 protein show impaired ciliary mechanosensation, and this impairment has been proposed to promote cystogenesis. However, most cyst epithelia of human ADPKD kidneys appear to express full-length PC1 and PC2 in normal or increased abundance. We show that confluent primary ADPKD cyst cells with the novel PC1 mutation ΔL2433 and with normal abundance of PC1 and PC2 polypeptides lack ciliary PC1 and often lack ciliary PC2, whereas PC1 and PC2 are both present in cilia of confluent normal human kidney (NK) epithelial cells in primary culture. Confluent NK cells respond to shear stress with transient increases in cytoplasmic Ca2+ concentration ([Ca2+]i), dependent on both extracellular Ca2+ and release from intracellular stores. In contrast, ADPKD cyst cells lack flow-sensitive [Ca2+]i signaling and exhibit reduced endoplasmic reticulum Ca2+ stores and store-depletion-operated Ca2+ entry but retain near-normal [Ca2+]i responses to ANG II and to vasopressin. Expression of wild-type and mutant CD16.7-PKD1(115–226) fusion proteins reveals within the COOH-terminal 112 amino acids of PC1 a coiled-coil domain-independent ciliary localization signal. However, the coiled-coil domain is required for CD16.7-PKD1(115–226) expression to accelerate decay of the flow-induced Ca2+ signal in NK cells. These data provide evidence for ciliary dysfunction and polycystin mislocalization in human ADPKD cells with normal levels of PC1.

scholarly journals Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities

2016 ◽  
Vol 213 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Holly A. Ping ◽  
Lauren M. Kraft ◽  
WeiTing Chen ◽  
Amy E. Nilles ◽  
Laura L. Lackner
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Coiled Coil ◽  
Lipid Binding ◽  
General Mechanism ◽  
Cell Cortex ◽  
Pleckstrin Homology Domain ◽  
Binding Domains ◽  
Coiled Coil Domain ◽  
Mitochondrial Distribution

The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.

scholarly journals Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go

1987 ◽  
Vol 246 (2) ◽  
pp. 431-439 ◽  
Author(s):  
G L Waldo ◽  
T Evans ◽  
E D Fraser ◽  
J K Northup ◽  
M W Martin ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Binding Activity ◽  
Bovine Brain ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Gtp Binding ◽  
Guanine Nucleotide Binding ◽  
The Brain

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.

Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas

2000 ◽  
Vol 113 (11) ◽  
pp. 1963-1971 ◽  
Author(s):  
R.J. Finst ◽  
P.J. Kim ◽  
E.R. Griffis ◽  
L.M. Quarmby
Keyword(s):  
Coiled Coil ◽  
Subcellular Fractionation ◽  
Rt Pcr ◽  
Type Locus ◽  
Microtubule Severing ◽  
Calmodulin Binding ◽  
Amino Terminal ◽  
Binding Domains ◽  
Coiled Coil Domain ◽  
Novel Protein

A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca(2+)-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca(2+)-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca(2+)/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca(2+)-activated, Ca(2+)-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.

scholarly journals Functional Evolution of the Photolyase/Cryptochrome Protein Family: Importance of the C Terminus of Mammalian CRY1 for Circadian Core Oscillator Performance

2006 ◽  
Vol 26 (5) ◽  
pp. 1743-1753 ◽  
Author(s):  
Inês Chaves ◽  
Kazuhiro Yagita ◽  
Sander Barnhoorn ◽  
Hitoshi Okamura ◽  
Gijsbertus T. J. van der Horst ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Coiled Coil ◽  
Structural Similarity ◽  
Light Signaling ◽  
Core Domain ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Localization Signal ◽  
Species Specific

ABSTRACT Cryptochromes (CRYs) are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. Here, we show that the C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drive nuclear localization via two independent mechanisms and shift the equilibrium of shuttling mammalian CRY1 (mCRY1)/mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquirement of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family.

scholarly journals Functions of Gle1 are governed by two distinct modes of self-association

2020 ◽  
Author(s):  
Aaron C. Mason ◽  
Susan R. Wente
Keyword(s):  
Translation Initiation ◽  
Mrna Export ◽  
Coiled Coil ◽  
Stress Granule ◽  
Formation Mechanisms ◽  
Dependent Manner ◽  
Rna Helicases ◽  
Granule Formation ◽  
Coiled Coil Domain ◽  
Self Association

AbstractGle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate (IP6) binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel ten amino acid aggregation prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and non-stressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.

scholarly journals The GTP-binding Release Factor eRF3 as a Key Mediator Coupling Translation Termination to mRNA Decay

2004 ◽  
Vol 279 (44) ◽  
pp. 45693-45700 ◽  
Author(s):  
Tetsuo Kobayashi ◽  
Yuji Funakoshi ◽  
Shin-ichi Hoshino ◽  
Toshiaki Katada
Keyword(s):  
Mrna Decay ◽  
Translation Termination ◽  
Binding Protein ◽  
Binding Activity ◽  
Release Factor ◽  
Guanine Nucleotide Binding Protein ◽  
Eukaryotic Translation ◽  
Gtp Binding ◽  
Nonsense Mediated Mrna Decay ◽  
Guanine Nucleotide Binding

GTP is essential for eukaryotic translation termination, where the release factor 3 (eRF3) complexed with eRF1 is involved as the guanine nucleotide-binding protein. In addition, eRF3 regulates the termination-coupled events, eRF3 interacts with poly(A)-binding protein (Pab1) and the surveillance factor Upf1 to mediate normal and nonsense-mediated mRNA decay. However, the roles of GTP binding to eRF3 in these processes remain largely unknown. Here, we showed in yeast that GTP is essentially required for the association of eRF3 with eRF1, but not with Pab1 and Upf1. A mutation in the GTP-binding motifs of eRF3 impairs the eRF1-binding ability without altering the Pab1- or Upf1-binding activity. Interestingly, the mutation causes not only a defect in translation termination but also delay of normal and nonsense-mediated mRNA decay, suggesting that GTP/eRF3-dependent termination exerts its influence on the subsequent mRNA degradation. The termination reaction itself is not sufficient, but eRF3 is essential for triggering mRNA decay. Thus, eRF3 is a key mediator that transduces termination signal to mRNA decay.

scholarly journals Subcellular Localization of RPB5-Mediating Protein and Its Putative Functional Partner

2004 ◽  
Vol 24 (19) ◽  
pp. 8556-8566 ◽  
Author(s):  
Luvsanjav Delgermaa ◽  
Naoyuki Hayashi ◽  
Dorjbal Dorjsuren ◽  
Takahiro Nomura ◽  
Le Thi-Thu Thuy ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Nuclear Localization ◽  
Dna Methyltransferase ◽  
Fluorescent Protein ◽  
Coiled Coil ◽  
Cellular Protein ◽  
Dependent Manner ◽  
Tor Signaling ◽  
Localization Signal ◽  
Green Fluorescent

ABSTRACT We previously identified a novel cellular protein, RPB5-mediating protein (RMP), that retains corepressor activity and functionally antagonizes transcriptional modulation via hepatitis B virus X protein. The subcellular localization of RMP was examined using green fluorescent protein-fused protein forms. We found that a nuclear localization signal (NLS) and a coiled-coil (CC) domain functioning as a cytoplasmic localization signal (CLS) are important for the subcellular localization of RMP. The CLS apparently acts dominantly, since RMP was mostly localized in the cytoplasm with weak and diffuse signals in the nucleus, and the NLS was indispensable for the nuclear localization of RMP only in the absence of the CLS. Using a yeast two-hybrid method, we isolated a putative corepressor, DNA methyltransferase 1-associating protein (DMAP1), which was found to bind to the CC domain of RMP. DMAP1 facilitated the nuclear localization of RMP and the corepressor activity of RMP in a dose-dependent manner by interacting with the CC domain of RMP. These results are discussed in light of a recent paper showing a novel evolutionarily conserved role of URI in the TOR signaling pathway.

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