scholarly journals PRELI (protein of relevant evolutionary and lymphoid interest) is located within an evolutionarily conserved gene cluster on chromosome 5q34-q35 and encodes a novel mitochondrial protein

2004 ◽  
Vol 378 (3) ◽  
pp. 817-825 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Sally A. STUBBS ◽  
Jimmy KYAW TUN ◽  
Jack P. LEEK ◽  
Alexander F. MARKHAM ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Subcellular Location ◽  
Protein Fusion ◽  
Evolutionarily Conserved ◽  
N Terminus ◽  
Targeting Signal ◽  
Conserved Gene ◽  
Green Fluorescent

The characterization of mitochondrial proteins is important for the understanding of both normal cellular function and mitochondrial disease. In the present study we identify a novel mitochondrial protein, PRELI (protein of relevant evolutionary and lymphoid interest), that is encoded within the evolutionarily conserved MAD3/PRELI/RAB24 gene cluster located at chromosome 5q34–q35. Mouse Preli is expressed at high levels in all settings analysed; it is co-expressed with Rab24 from a strong bi-directional promoter, and is regulated independently from the S-phase-specific Mad3 gene located at its 3´ end. PRELI contains a stand-alone 170 amino acid PRELI/MSF1p´ motif at its N-terminus. This domain is found in a variety of proteins from diverse eukaryotes including yeast, Drosophila and mammals, but its function is unknown, and the subcellular location of higher eukaryotic PRELI/MSF1P´ proteins has not been determined previously. We show here that PRELI is located in the mitochondria, and by using green-fluorescent-protein fusion proteins we identify a mitochondrial targeting signal at its N-terminus.

scholarly journals Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

1997 ◽  
Vol 17 (9) ◽  
pp. 5001-5015 ◽  
Author(s):  
N I Zanchin ◽  
P Roberts ◽  
A DeSilva ◽  
F Sherman ◽  
D S Goldfarb
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Acid Protein ◽  
Conserved Gene ◽  
Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

scholarly journals Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

2009 ◽  
Vol 191 (9) ◽  
pp. 3149-3161 ◽  
Author(s):  
Suzanne R. Lybarger ◽  
Tanya L. Johnson ◽  
Miranda D. Gray ◽  
Aleksandra E. Sikora ◽  
Maria Sandkvist
Keyword(s):  
Vibrio Cholerae ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Outer Membrane Proteins ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Protein Fusion ◽  
Green Fluorescent

ABSTRACT Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

scholarly journals Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

2009 ◽  
Vol 192 (1) ◽  
pp. 38-45 ◽  
Author(s):  
Melanie B. Berkmen ◽  
Catherine A. Lee ◽  
Emma-Kate Loveday ◽  
Alan D. Grossman
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Donor Cell ◽  
Subcellular Location ◽  
Conjugative Transfer ◽  
Protein Fusion ◽  
Cell Pole ◽  
A Cell ◽  
Green Fluorescent ◽  
Integrative And Conjugative Element

ABSTRACT ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that ICEBs1 affects the subcellular position of the replisome.

scholarly journals A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria

2004 ◽  
Vol 382 (1) ◽  
pp. 385-392 ◽  
Author(s):  
Abhijit MUKHOPADHYAY ◽  
Li NI ◽  
Henry WEINER
Keyword(s):  
Signal Peptide ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Major Pathway ◽  
Native Proteins ◽  
C Terminus ◽  
N Terminus ◽  
Green Fluorescent

The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDHsp–DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDHsp–DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.

scholarly journals The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1377
Author(s):  
Adriano M. Gigante ◽  
Francisco Olivença ◽  
Maria João Catalão ◽  
Paula Leandro ◽  
José Moniz-Pereira ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Structural Similarity ◽  
Lytic Cycle ◽  
Specific Lysis ◽  
Double Stranded Dna ◽  
The Family ◽  
N Terminus ◽  
Green Fluorescent

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

scholarly journals The fungal gene cluster for biosynthesis of the antibacterial agent viriditoxin

2019 ◽  
Author(s):  
Andrew S. Urquhart ◽  
Jinyu Hu ◽  
Yit-Heng Chooi ◽  
Alexander Idnurm
Keyword(s):  
Secondary Metabolites ◽  
Gene Cluster ◽  
Gene Disruption ◽  
Biosynthetic Pathway ◽  
Fluorescent Protein ◽  
Model Species ◽  
Paecilomyces Variotii ◽  
Bioinformatic Approaches ◽  
Green Fluorescent ◽  
Fungal Gene

AbstractBackgroundViriditoxin is one of the ‘classical’ secondary metabolites produced by fungi and that has antibacterial and other activities; however, the mechanism of its biosynthesis has remained unknown.ResultsHere, a gene cluster responsible for its synthesis was identified, using bioinformatic approaches from two species that produce viriditoxin and then through gene disruption and metabolite profiling. All eight genes in the cluster inPaecilomyces variotiiwere mutated, revealing their roles in the synthesis of this molecule and establishing its biosynthetic pathway which includes an interesting Baeyer-Villiger monooxygenase catalyzed reaction. Additionally, a candidate catalytically-inactive hydrolase was identified as being required for the stereoselective biosynthesis of (M)-viriditoxin. The localization of two proteins were assessed by fusing these proteins to green fluorescent protein, revealing that at least two intracellular structures are involved in the compartmentalization of the synthesis steps of this metabolite.ConclusionsThe full pathway for synthesis of viriditoxin was established by a combination of genomics, bioinformatics, gene disruption and chemical analysis processes. Hence, this work reveals the basis for the synthesis of an understudied class of fungal secondary metabolites and provides a new model species for understanding the synthesis of biaryl compounds with a chiral axis.

scholarly journals Green Fluorescent Chimeras Indicate Nonpolar Localization of Pullulanase Secreton Components PulL and PulM

2006 ◽  
Vol 188 (8) ◽  
pp. 2928-2935 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Olivera Francetic ◽  
Anthony P. Pugsley
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Klebsiella Oxytoca ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Native Proteins ◽  
Type Ii Secretion System ◽  
Green Fluorescent

ABSTRACT The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.

A novel linker histone-like protein is associated with cytoplasmic filaments inCaenorhabditis elegans

2002 ◽  
Vol 115 (14) ◽  
pp. 2881-2891
Author(s):  
Monika A. Jedrusik ◽  
Stefan Vogt ◽  
Peter Claus ◽  
Ekkehard Schulze
Keyword(s):  
Rna Interference ◽  
Fluorescent Protein ◽  
Muscle Growth ◽  
Egg Laying ◽  
Globular Domain ◽  
Protein Fusion ◽  
Linker Histones ◽  
C Elegans ◽  
Fusion Protein Expression ◽  
Green Fluorescent

The histone H1 complement of Caenorhabditis elegans contains a single unusual protein, H1.X. Although H1.X possesses the globular domain and the canonical three-domain structure of linker histones, the amino acid composition of H1.X is distinctly different from conventional linker histones in both terminal domains. We have characterized H1.X in C. elegans by antibody labeling, green fluorescent protein fusion protein expression and RNA interference. Unlike normal linker histones, H1.X is a cytoplasmic as well as a nuclear protein and is not associated with chromosomes. H1.X is most prominently expressed in the marginal cells of the pharynx and is associated with a peculiar cytoplasmic cytoskeletal structure therein, the tonofilaments. Additionally H1.X::GFP is expressed in the cytoplasm of body and vulva muscle cells, neurons, excretory cells and in the nucleoli of embryonic blastomeres and adult gut cells. RNA interference with H1.X results in uncoordinated and egg laying defective animals, as well as in a longitudinally enlarged pharynx. These phenotypes indicate a cytoplasmic role of H1.X in muscle growth and muscle function.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

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