scholarly journals Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii

Genes ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 434
Author(s):  
Alison Mbekeani ◽  
Will Stanley ◽  
Vishal Kalel ◽  
Noa Dahan ◽  
Einat Zalckvar ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Metabolic Energy ◽  
Peroxisomal Protein ◽  
Genes Encoding ◽  
Peroxisomal Targeting ◽  
Membrane Bound ◽  
Green Fluorescent

Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids—which subsequently provide an important source of metabolic energy—and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian Toxoplasma gondii was shown to rescue the import defect of Pex5-deleted Saccharomyces cerevisiae. In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Toxoplasma. Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised Toxoplasma sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5–PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.

Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system

2000 ◽  
Vol 113 (22) ◽  
pp. 3969-3977 ◽  
Author(s):  
A. DeRocher ◽  
C.B. Hagen ◽  
J.E. Froehlich ◽  
J.E. Feagin ◽  
M. Parsons
Keyword(s):  
Green Fluorescent Protein ◽  
Toxoplasma Gondii ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Transit Peptide ◽  
Green Fluorescent ◽  
Secretory System

Apicomplexan parasites possess a plastid-like organelle called the apicoplast. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nucleus and imported post-translationally. T. gondii apicoplast proteins often have a long N-terminal extension that directs the protein to the apicoplast. It can be modeled as a bipartite targeting sequence that contains a signal sequence and a plastid transit peptide. We identified two nuclearly encoded predicted plastid proteins and made fusions with green fluorescent protein to study protein domains required for apicoplast targeting. The N-terminal 42 amino acids of the apicoplast ribosomal protein S9 directs secretion of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to direct green fluorescent protein to the apicoplast. The predicted transit peptide domain of the S9 targeting sequence directs protein to the mitochondrion in vivo. The transit peptide can also direct import of green fluorescent protein into chloroplasts in vitro. These data substantiate the model that protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import.

scholarly journals Peroxisome Function Is Required for Virulence and Survival of Fusarium graminearum

2012 ◽  
Vol 25 (12) ◽  
pp. 1617-1627 ◽  
Author(s):  
Kyunghun Min ◽  
Hokyoung Son ◽  
Jungkwan Lee ◽  
Gyung Ja Choi ◽  
Jin-Cheol Kim ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fusarium Graminearum ◽  
Protein Import ◽  
Fluorescent Proteins ◽  
Deletion Mutants ◽  
In Planta ◽  
Peroxisomal Protein ◽  
Peroxisomal Targeting ◽  
Signal Type

Peroxisomes are organelles that are involved in a number of important cellular metabolic processes, including the β-oxidation of fatty acids, biosynthesis of secondary metabolites, and detoxification of reactive oxygen species (ROS). In this study, the role of peroxisomes was examined in Fusarium graminearum by targeted deletion of three genes (PEX5, PEX6, and PEX7) encoding peroxin (PEX) proteins required for peroxisomal protein import. PEX5 and PEX7 deletion mutants were unable to localize the fluorescently tagged peroxisomal targeting signal type 1 (PTS1)- and PTS2-containing proteins to peroxisomes, respectively, whereas the PEX6 mutant failed to localize both fluorescent proteins. Deletion of PEX5 and PEX6 resulted in retarded growth on long-chain fatty acids and butyrate, while the PEX7 deletion mutants utilized fatty acids other than butyrate. Virulence on wheat heads was greatly reduced in the PEX5 and PEX6 deletion mutants, and they were defective in spreading from inoculated florets to the adjacent spikelets through rachis. Deletion of PEX5 and PEX6 dropped survivability of aged cells in planta and in vitro due to the accumulation of ROS followed by necrotic cell death. These results demonstrate that PTS1-dependent peroxisomal protein import mediated by PEX5 and PEX6 are critical to virulence and survival of F. graminearum.

scholarly journals The peroxisomal protein import machinery displays a preference for monomeric substrates

Open Biology ◽  
2015 ◽  
Vol 5 (4) ◽  
pp. 140236 ◽  
Author(s):  
Marta O. Freitas ◽  
Tânia Francisco ◽  
Tony A. Rodrigues ◽  
Celien Lismont ◽  
Pedro Domingues ◽  
...  
Keyword(s):  
Protein Import ◽  
Urate Oxidase ◽  
Experimental Conditions ◽  
Peroxisomal Membrane ◽  
Oligomeric Proteins ◽  
Peroxisomal Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Monomeric Proteins

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.

scholarly journals Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders.

1995 ◽  
Vol 130 (1) ◽  
pp. 51-65 ◽  
Author(s):  
E A Wiemer ◽  
W M Nuttley ◽  
B L Bertolaet ◽  
X Li ◽  
U Francke ◽  
...  
Keyword(s):  
Protein Import ◽  
Zellweger Syndrome ◽  
Complementation Group ◽  
Cell System ◽  
Peroxisomal Disorders ◽  
Peroxisomal Membrane ◽  
Peroxisomal Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal

Two peroxisomal targeting signals, PTS1 and PTS2, are involved in the import of proteins into the peroxisome matrix. Human patients with fatal generalized peroxisomal deficiency disorders fall into at least nine genetic complementation groups. Cells from many of these patients are deficient in the import of PTS1-containing proteins, but the causes of the protein-import defect in these patients are unknown. We have cloned and sequenced the human cDNA homologue (PTS1R) of the Pichia pastoris PAS8 gene, the PTS1 receptor (McCollum, D., E. Monosov, and S. Subramani. 1993. J. Cell Biol. 121:761-774). The PTS1R mRNA is expressed in all human tissues examined. Antibodies to the human PTS1R recognize this protein in human, monkey, rat, and hamster cells. The protein is localized mainly in the cytosol but is also found to be associated with peroxisomes. Part of the peroxisomal PTS1R protein is tightly bound to the peroxisomal membrane. Antibodies to PTS1R inhibit peroxisomal protein-import of PTS1-containing proteins in a permeabilized CHO cell system. In vitro-translated PTS1R protein specifically binds a serine-lysine-leucine-peptide. A PAS8-PTS1R fusion protein complements the P. pastoris pas8 mutant. The PTS1R cDNA also complements the PTS1 protein-import defect in skin fibroblasts from patients--belonging to complementation group two--diagnosed as having neonatal adrenoleukodystrophy or Zellweger syndrome. The PTS1R gene has been localized to a chromosomal location where no other peroxisomal disorder genes are known to map. Our findings represent the only case in which the molecular basis of the protein-import deficiency in human peroxisomal disorders is understood.

scholarly journals Isolation of Human Small Extracellular Vesicles and Tracking of Their Uptake by Retinal Pigment Epithelial Cells In Vitro

2020 ◽  
Vol 21 (11) ◽  
pp. 3799
Author(s):  
Irene C. Marcu ◽  
Naja Eberhard ◽  
Anaïs Yerly ◽  
Verena Balmer ◽  
Andrew Hemphill ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fluorescent Protein ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Target Cells ◽  
Rpe Cells ◽  
Membrane Bound ◽  
Green Fluorescent

Small extracellular vesicles (EVs) are among the most frequently investigated EVs and play major roles in intercellular communication by delivering various cargo molecules to target cells. They could potentially represent an alternative delivery strategy to treat ocular toxoplasmosis, a parasitosis affecting the retinal pigment epithelium (RPE). To date, the uptake of human small EVs by RPE cells has never been reported. In this study, we report on the intracellular uptake of fluorescently labelled human urine and fibroblast-derived small EVs by human RPE cells. In summary, both dye-labelled urinary small EVs and small EVs obtained from fibroblasts stably expressing membrane-bound green fluorescent protein were successfully internalized by RPE cells as revealed by immunohistochemistry. In recipient ARPE19 cells, BODIPY-labelled small EVs were found in close vicinity to the parasite Toxoplasma gondii. Additionally, an ultrastructural method was enabled to distinguish between labelled exogenous and endogenous small EVs within target cells.

scholarly journals Liquid-liquid phase separation of florigen activation complex induces flowering

2021 ◽  
Author(s):  
Ken-ichiro Taoka ◽  
Hiroyuki Tsuji ◽  
Suai Anzawa ◽  
Mayu Enomoto ◽  
Yuka Koizumi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Leucine Zipper ◽  
Fluorescent Protein ◽  
Gene Activation ◽  
Heading Date ◽  
Dependent Manner ◽  
Basic Leucine Zipper ◽  
Genes Encoding ◽  
Green Fluorescent

Floral transition, regulated by the systemic action of the mobile florigen protein FLOWERING LOCUS T (FT), is essential for successful plant reproduction1. How FT controls downstream gene expression remains incompletely understood, although it relies on the florigen activation complex (FAC), a core component of FT function2-4. The FAC is a nucleus-localized transcriptional activator of genes encoding MADS-box transcription factors critical to reproductive development and consists of florigen FT; a scaffold 14-3-3 protein that is a key component for complex assembly; and FD, a basic leucine-zipper protein that recruits the FAC to DNA. Here we report that the FAC exhibits phase separation. In rice shoot apical meristem cells, rice florigen Heading date 3a (Hd3a) fused to the green fluorescent protein formed speckles in the nucleus. The FAC speckle is formed in a FAC-dependent manner in tobacco cells. Recombinant Hd3a, but not OsFD1, phase-separated in vitro, and this effect was enhanced in the presence of 14-3-3 protein. Furthermore, mutations affecting functionally important residues in the pocket region or C-terminal disordered region of Hd3a affected FAC phase separation, providing a biochemical framework for the protein's effect on flowering. The ability to form condensates via phase transition represents a previously unknown mechanism for gene activation by the FAC.

scholarly journals Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein

2003 ◽  
Vol 374 (2) ◽  
pp. 329-335 ◽  
Author(s):  
Salama R. ABDELRAHEIM ◽  
David G. SPILLER ◽  
Alexander G. McLENNAN
Keyword(s):  
Fluorescent Protein ◽  
Nudix Hydrolase ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Diadenosine Triphosphate ◽  
Green Fluorescent ◽  
Cytoplasmic Structures

The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2′,5′-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)+, ADP-ribose and diadenosine triphosphate. Km values for NADH, NADPH and NAD+ are 11, 16 and 190 μM and kcat values are 11, 16 and 10.5 s−1 respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 μM Mn2+ ions in vitro, with 3-fold lower activity at 0.4 mM Mg2+. Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.

scholarly journals Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Antonia Werner ◽  
Kolja L. Otte ◽  
Gertrud Stahlhut ◽  
Stefanie Pöggeler
Keyword(s):  
Cell Biology ◽  
Cell Imaging ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Mycelial Fungi ◽  
Filamentous Ascomycete ◽  
Bright Fluorescence ◽  
Peroxisomal Targeting ◽  
Branchiostoma Lanceolatum ◽  
Green Fluorescent

AbstractThe engineered monomeric version of the lancelet Branchiostoma lanceolatum fluorescent protein, mNeonGreen (mNG), has several positive characteristics, such as a very bright fluorescence, high photostability and fast maturation. These features make it a good candidate for the utilization as fluorescent tool for cell biology and biochemical applications in filamentous fungi. We report the generation of plasmids for the expression of the heterologous mNG gene under the control of an inducible and a constitutive promoter in the filamentous ascomycete Sordaria macrospora and display a stable expression of mNG in the cytoplasm. To demonstrate its usefulness for labeling of organelles, the peroxisomal targeting sequence serine-lysine-leucine (SKL) was fused to mNG. Expression of this tagged version led to protein import of mNG into peroxisomes and their bright fluorescence in life cell imaging.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

Sign in / Sign up

Export Citation Format

Share Document