scholarly journals Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

2004 ◽  
Vol 3 (3) ◽  
pp. 663-674 ◽  
Author(s):  
Omar S. Harb ◽  
Bithi Chatterjee ◽  
Martin J. Fraunholz ◽  
Michael J. Crawford ◽  
Manami Nishi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Parasitophorous Vacuole ◽  
Transit Peptide ◽  
Functional Domains ◽  
Transit Peptides ◽  
Endosymbiotic Organelle

ABSTRACT Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle—the apicoplast—acquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP+ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments.

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 Protein Targeting to the Parasitophorous Vacuole Membrane of Plasmodium falciparum

2011 ◽  
Vol 10 (6) ◽  
pp. 744-752 ◽  
Author(s):  
Saliha Eksi ◽  
Kim C. Williamson
Keyword(s):  
Amino Acids ◽  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Parasitophorous Vacuole ◽  
Reporter Protein ◽  
Content Type ◽  
Gfp Reporter

ABSTRACTRed blood cell (RBC) invasion and parasitophorous vacuole (PV) formation byPlasmodium falciparumare critical for the development and pathogenesis of malaria, a continuing global health problem. Expansion of the PV membrane (PVM) during growth is orchestrated by the parasite. This is particularly important in mature RBCs, which lack internal organelles and no longer actively synthesize membranes. Pfs16, a 16-kDa integral PVM protein expressed by gametocytes, was chosen as a model for studying the trafficking of material from the parasite across the PV space to the PVM. The locations of Pfs16-green fluorescent protein (GFP) reporter proteins containing distinct regions of Pfs16 were tracked from RBC invasion to emergence. Inclusion of the 53 C-terminal amino acids (aa) of Pfs16 to a GFP reporter construct already containing the N-terminal secretory signal sequence was sufficient for targeting to and retention on the PVM. An amino acid motif identified in this region was also found in seven other known PVM proteins. Removal of the 11 C-terminal aa did not affect PVM targeting, but membrane retention was decreased. Additionally, during emergence from the PVM and RBC, native Pfs16 and the full-length Pfs16-GFP reporter protein were found to concentrate on the ends of the gametocyte. Capping was not observed in constructs lacking the amino acids between the N-terminal secretory signal sequence and the transmembrane domain, suggesting that this region, which is not required for PVM targeting, is involved in capping. This is the first report to define the amino acid domains required for targeting to theP. falciparumPVM.

scholarly journals The Equine Herpesvirus 1 US2 Homolog Encodes a Nonessential Membrane-Associated Virion Component

1999 ◽  
Vol 73 (4) ◽  
pp. 3430-3437 ◽  
Author(s):  
Alexandra Meindl ◽  
Nikolaus Osterrieder
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Viral Envelope ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Amino Terminal ◽  
Infected Cells ◽  
Herpesvirus 1

ABSTRACT Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 US2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 US2 protein specifically detected a protein with an M r of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-M r Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-M r Us2 polypeptide. Irrespective of its size, the US2 protein was incorporated into virions. The EHV-1 US2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 US2 protein or to a truncated US2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 US2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the US2 protein in the viral envelope and plasma membrane of infected cells, a US2-negative RacL11 mutant (L11ΔUS2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a US2-repaired virus. After infection of BALB/c mice with L11ΔUS2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 US2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.

scholarly journals Manipulating sorting signals to generate co-expression of somatostatin and eGFP in the regulated secretory pathway from a monocistronic construct

2004 ◽  
Vol 33 (2) ◽  
pp. 523-532 ◽  
Author(s):  
J S Davies ◽  
J L Holter ◽  
D Knight ◽  
S M Beaucourt ◽  
D Murphy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Proteolytic Processing ◽  
Biologically Active ◽  
Foreign Protein ◽  
Entry Site ◽  
Fluorescent Markers ◽  
Regulated Secretory Pathway ◽  
Neuroendocrine Systems

Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.

scholarly journals The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane

2001 ◽  
Vol 154 (1) ◽  
pp. 95-108 ◽  
Author(s):  
Anthony P. Sinai ◽  
Keith A. Joiner
Keyword(s):  
Toxoplasma Gondii ◽  
Fluorescent Protein ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
In Vitro Assays ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Targeting ◽  
Parasitophorous Vacuole Membrane ◽  
Parasite Protein ◽  
Vacuole Membrane

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM–organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH2-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30–amino acid (aa) NH2-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH2-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

‘Piggy-Back’ Transport of Xenopus Hyaluronan Synthase (XHAS1) via the Secretory Pathway to the Plasma Membrane

2003 ◽  
Vol 384 (1) ◽  
pp. 175-182 ◽  
Author(s):  
J. Müllegger ◽  
A. Rustom ◽  
G. Kreil ◽  
H.-H. Gerdes ◽  
G. Lepperdinger
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
De Novo ◽  
Signal Sequence ◽  
Vero Cells ◽  
In Vitro Translation ◽  
Hyaluronan Synthase ◽  
Microsomal Membranes

AbstractHyaluronan is the sole glycosaminoglycan whose biosynthesis takes place directly at the plasma membrane. The mechanism by which hyaluronan synthase (HAS) becomes inserted there, as well as the question of how the enzyme discriminates between particular membrane species in polarized cells, are largely unknown. In vitro translation of HAS suggested that the nascent protein becomes stabilized in the presence of microsomal membranes, but would not insert spontaneously into membranes after being translated in the absence of those. We therefore monitored the membrane attachment of enzymatically active fusion proteins consisting of Xenopus HAS1 and green fluorescent protein shortly after de novo synthesis in Vero cells. Our data strongly suggest that HAS proteins are directly translated on the ER membrane without exhibiting an N-terminal signal sequence. From there the inactive protein is transferred to the plasma membrane via the secretory pathway. For unknown reasons, HAS inserted into membranes other than the plasma membrane remains inactive.

scholarly journals Correct Promoter Control Is Needed for Trafficking of the Ring-Infected Erythrocyte Surface Antigen to the Host Cytosol in Transfected Malaria Parasites

2004 ◽  
Vol 72 (10) ◽  
pp. 6095-6105 ◽  
Author(s):  
Melanie Rug ◽  
Mark E. Wickham ◽  
Michael Foley ◽  
Alan F. Cowman ◽  
Leann Tilley
Keyword(s):  
Surface Antigen ◽  
Transgene Expression ◽  
Infected Erythrocyte ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Parasitophorous Vacuole ◽  
Chimeric Protein ◽  
Terminal Region ◽  
Signal Sequences ◽  
Erythrocyte Surface

ABSTRACT Following invasion of human erythrocytes, the malaria parasite, Plasmodium falciparum, exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the P. falciparum genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized; however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the P. falciparum ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependant on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the RESA promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.

scholarly journals The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
William J. Blakely ◽  
Michael J. Holmes ◽  
Gustavo Arrizabalaga
Keyword(s):  
Acid Phosphatase ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Secretory Pathway ◽  
Parasitophorous Vacuole ◽  
Severe Disease ◽  
Immune Recognition ◽  
Phosphatase Domain ◽  
Content Type ◽  
Cell Defense

ABSTRACT During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parasitophorous vacuole to safely reside within the cell, while it is partitioned from host cell defense mechanisms. From within this safe niche, parasites sabotage multiple host cell systems, including gene expression, apoptosis, and intracellular immune recognition, by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. The translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes the proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined that GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins, including MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell c-Myc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen. IMPORTANCE Approximately one-third of humans are infected with the parasite Toxoplasma gondii. Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. Toxoplasma replication requires that it enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

scholarly journals Analysis of Euglena gracilis Plastid-Targeted Proteins Reveals Different Classes of Transit Sequences

2006 ◽  
Vol 5 (12) ◽  
pp. 2079-2091 ◽  
Author(s):  
Dion G. Durnford ◽  
Michael W. Gray
Keyword(s):  
Euglena Gracilis ◽  
Protein Targeting ◽  
Expressed Sequence Tag ◽  
Signal Sequence ◽  
Transit Peptide ◽  
Hydrophobic Domain ◽  
Transit Peptides ◽  
Endosymbiotic Event ◽  
Plastid Protein ◽  
Plastid Transit Peptide

ABSTRACT The plastid of Euglena gracilis was acquired secondarily through an endosymbiotic event with a eukaryotic green alga, and as a result, it is surrounded by a third membrane. This membrane complexity raises the question of how the plastid proteins are targeted to and imported into the organelle. To further explore plastid protein targeting in Euglena, we screened a total of 9,461 expressed sequence tag (EST) clusters (derived from 19,013 individual ESTs) for full-length proteins that are plastid localized to characterize their targeting sequences and to infer potential modes of translocation. Of the 117 proteins identified as being potentially plastid localized whose N-terminal targeting sequences could be inferred, 83 were unique and could be classified into two major groups. Class I proteins have tripartite targeting sequences, comprising (in order) an N-terminal signal sequence, a plastid transit peptide domain, and a predicted stop-transfer sequence. Within this class of proteins are the lumen-targeted proteins (class IB), which have an additional hydrophobic domain similar to a signal sequence and required for further targeting across the thylakoid membrane. Class II proteins lack the putative stop-transfer sequence and possess only a signal sequence at the N terminus, followed by what, in amino acid composition, resembles a plastid transit peptide. Unexpectedly, a few unrelated plastid-targeted proteins exhibit highly similar transit sequences, implying either a recent swapping of these domains or a conserved function. This work represents the most comprehensive description to date of transit peptides in Euglena and hints at the complex routes of plastid targeting that must exist in this organism.

scholarly journals Dually localized proteins found in both the apicoplast and mitochondrion utilize the Golgi-dependent pathway for apicoplast targeting in Toxoplasma gondii

2020 ◽  
Author(s):  
Aparna Prasad ◽  
Pragati Mastud ◽  
Swati Patankar
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Apicomplexan Parasites ◽  
Evolutionary Origins ◽  
Molecular Features ◽  
Experimental Approaches ◽  
Endosymbiotic Organelle ◽  
First Time ◽  
Dependent Pathway

ABSTRACTLike other apicomplexan parasites, Toxoplasma gondii harbours a four-membraned endosymbiotic organelle - the apicoplast. Apicoplast proteins are nuclear-encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell’s secretory pathway involving the Golgi, while the other is a unique Golgi-independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi-independent pathway, while a handful of reports show that a few proteins use the Golgi-dependent pathway. This has led to an emphasis towards the unique Golgi-independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known. In this report, we systematically test eight apicoplast proteins, using a C-terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN) are trafficked through the Golgi while proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN suggested that the evolutionary origins of TgSOD2, TgTPx1/2 lie in the mitochondrion while TgACN appears to have originated from the apicoplast. Collectively, with these results, for the first time, we establish that the driver of the Golgi-dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.

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