scholarly journals Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

2013 ◽  
Vol 13 (3) ◽  
pp. 353-362 ◽  
Author(s):  
Julie Kovářová ◽  
Eva Horáková ◽  
Piya Changmai ◽  
Marie Vancová ◽  
Julius Lukeš
Keyword(s):  
Trypanosoma Brucei ◽  
Morphological Changes ◽  
Scaffold Protein ◽  
Complex Life Cycle ◽  
Insect Vector ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
Dual Localization ◽  
Localization Signal

ABSTRACT Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

scholarly journals Mutation in the Fe–S scaffold protein Isu bypasses frataxin deletion

2011 ◽  
Vol 441 (1) ◽  
pp. 473-480 ◽  
Author(s):  
Heeyong Yoon ◽  
Ramesh Golla ◽  
Emmanuel Lesuisse ◽  
Jayashree Pain ◽  
Jason E. Donald ◽  
...  
Keyword(s):  
Unique Feature ◽  
Mitochondrial Protein ◽  
Scaffold Protein ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Cluster Assembly ◽  
Iron Availability ◽  
Direct Effects ◽  
Cysteine Desulfurase ◽  
Mitochondrial Iron

Frataxin is a conserved mitochondrial protein deficient in patients with Friedreich's ataxia. Frataxin has been implicated in control of iron homoeostasis and Fe–S cluster assembly. In yeast or human mitochondria, frataxin interacts with components of the Fe–S cluster synthesis machinery, including the cysteine desulfurase Nfs1, accessory protein Isd11 and scaffold protein Isu. In the present paper, we report that a single amino acid substitution (methionine to isoleucine) at position 107 in the mature form of Isu1 restored many deficient functions in Δyfh1 or frataxin-depleted yeast cells. Iron homoeostasis was improved such that soluble/usable mitochondrial iron was increased and accumulation of insoluble/non-usable iron within mitochondria was largely prevented. Cytochromes were returned to normal and haem synthesis was restored. In mitochondria carrying the mutant Isu1 and no frataxin, Fe–S cluster enzyme activities were improved. The efficiency of new Fe–S cluster synthesis in isolated mitochondria was markedly increased compared with frataxin-negative cells, although the response to added iron was minimal. The M107I substitution in the highly conserved Isu scaffold protein is typically found in bacterial orthologues, suggesting that a unique feature of the bacterial Fe–S cluster machinery may be involved. The mechanism by which the mutant Isu bypasses the absence of frataxin remains to be determined, but could be related to direct effects on Fe–S cluster assembly and/or indirect effects on mitochondrial iron availability.

scholarly journals A Tale of Three Species: Adaptation ofSodalis glossinidiusto Tsetse Biology,WigglesworthiaMetabolism, and Host Diet

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Rebecca J. Hall ◽  
Lindsey A. Flanagan ◽  
Michael J. Bottery ◽  
Vicki Springthorpe ◽  
Stephen Thorpe ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Strong Dependence ◽  
Tsetse Fly ◽  
Disease Spread ◽  
Growth Media ◽  
Insect Vector ◽  
African Trypanosomiasis ◽  
Content Type ◽  
Sodalis Glossinidius

ABSTRACTThe tsetse fly is the insect vector for theTrypanosoma bruceiparasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium,Sodalis glossinidius. The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model ofS. glossinidiusmetabolism. The model enabled the design and experimental verification of a defined medium that supportsS. glossinidiusgrowthex vivo. This has been used subsequently to analyzein vitroaspects ofS. glossinidiusmetabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomerN-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence onl-glutamate as a source of carbon and nitrogen and by the ability to rescue a predictedl-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiontWigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution ofS. glossinidiusto exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibitS. glossinidiusgrowth and aid the reduction of trypanosomal transmission.IMPORTANCEHuman African trypanosomiasis is caused by theTrypanosoma bruceiparasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential forT. bruceilife cycle progression and transmission. The tsetse’s mutualistic endosymbiontSodalis glossinidiushas a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model ofS. glossinidiusmetabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects ofS. glossinidiusmetabolism. We presentS. glossinidiusas uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally,S. glossinidiushas adapted to the tsetse’s obligate symbiontWigglesworthia glossinidiaby scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission.

scholarly journals Frataxin-bypassing Isu1: characterization of the bypass activity in cells and mitochondria

2014 ◽  
Vol 459 (1) ◽  
pp. 71-81 ◽  
Author(s):  
Heeyong Yoon ◽  
Simon A. B. Knight ◽  
Alok Pandey ◽  
Jayashree Pain ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Scaffold Protein ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
In Cells

The Fe–S cluster assembly scaffold protein Isu1 with the amino acid change M107I is able to bypass lack of the frataxin by stimulating cysteine desulfurase activity in mitochondria, although a kinetic defect in Fe–S cluster assembly persists.

scholarly journals Overlapping Binding Sites of the Frataxin Homologue Assembly Factor and the Heat Shock Protein 70 Transfer Factor on the Isu Iron-Sulfur Cluster Scaffold Protein

2014 ◽  
Vol 289 (44) ◽  
pp. 30268-30278 ◽  
Author(s):  
Mateusz Manicki ◽  
Julia Majewska ◽  
Szymon Ciesielski ◽  
Brenda Schilke ◽  
Anna Blenska ◽  
...  
Keyword(s):  
Transfer Factor ◽  
Binding Sites ◽  
Scaffold Protein ◽  
Sequence Motif ◽  
Cluster Assembly ◽  
Assembly Process ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Cluster Transfer ◽  
Assembly Factor

In mitochondria FeS clusters, prosthetic groups critical for the activity of many proteins, are first assembled on Isu, a 14-kDa scaffold protein, and then transferred to recipient apoproteins. The assembly process involves interaction of Isu with both Nfs1, the cysteine desulfurase serving as a sulfur donor, and the yeast frataxin homolog (Yfh1) serving as a regulator of desulfurase activity and/or iron donor. Here, based on the results of biochemical experiments with purified wild-type and variant proteins, we report that interaction of Yfh1 with both Nfs1 and Isu are required for formation of a stable tripartite assembly complex. Disruption of either Yfh1-Isu or Nfs1-Isu interactions destabilizes the complex. Cluster transfer to recipient apoprotein is known to require the interaction of Isu with the J-protein/Hsp70 molecular chaperone pair, Jac1 and Ssq1. Here we show that the Yfh1 interaction with Isu involves the PVK sequence motif, which is also the site key for the interaction of Isu with Hsp70 Ssq1. Coupled with our previous observation that Nfs1 and Jac1 binding to Isu is mutually exclusive due to partially overlapping binding sites, we propose that such mutual exclusivity of cluster assembly factor (Nfs1/Yfh1) and cluster transfer factor (Jac1/Ssq1) binding to Isu has functional consequences for the transition from the assembly process to the transfer process, and thus regulation of the biogenesis of FeS cluster proteins.

scholarly journals Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

2011 ◽  
Vol 10 (6) ◽  
pp. 734-743 ◽  
Author(s):  
David F. Bruhn ◽  
Mark P. Sammartino ◽  
Michele M. Klingbeil
Keyword(s):  
Mitochondrial Dna ◽  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Dna Polymerases ◽  
Bloodstream Form ◽  
Complex Life Cycle ◽  
Replication Proteins ◽  
Kinetoplast Dna ◽  
Content Type ◽  
Parasite Viability

ABSTRACT Trypanosoma brucei , the causative agent of human African trypanosomiasis, has a complex life cycle that includes multiple life cycle stages and metabolic changes as the parasite switches between insect vector and mammalian host. The parasite's single mitochondrion contains a unique catenated mitochondrial DNA network called kinetoplast DNA (kDNA) that is composed of minicircles and maxicircles. Long-standing uncertainty about the requirement of kDNA in bloodstream form (BF) T. brucei has recently eroded, with reports of posttranscriptional editing and subsequent translation of kDNA-encoded transcripts as essential processes for BF parasites. These studies suggest that kDNA and its faithful replication are indispensable for this life cycle stage. Here we demonstrate that three kDNA replication proteins (mitochondrial DNA polymerases IB, IC, and ID) are required for BF parasite viability. Silencing of each polymerase was lethal, resulting in kDNA loss, persistence of prereplication DNA monomers, and collapse of the mitochondrial membrane potential. These data demonstrate that kDNA replication is indeed crucial for BF T. brucei . The contributions of mitochondrial DNA polymerases IB, IC, and ID to BF parasite viability suggest that these and other kDNA replication proteins warrant further investigation as a new class of targets for the development of antitrypanosomal drugs.

scholarly journals An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei

2014 ◽  
Vol 14 (2) ◽  
pp. 149-157 ◽  
Author(s):  
Natalie M. McAdams ◽  
Michelle L. Ammerman ◽  
Julee Nanduri ◽  
Kaylen Lott ◽  
John C. Fisk ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Rna Binding ◽  
Mitochondrial Gene ◽  
Mitochondrial Rna ◽  
Insect Vector ◽  
Sequence Specificity ◽  
Content Type

ABSTRACT In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo , inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei . TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.

scholarly journals Functional Analyses of Cytokinesis Regulators in Bloodstream Stage Trypanosoma brucei Parasites Identify Functions and Regulations Specific to the Life Cycle Stage

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Xuan Zhang ◽  
Tai An ◽  
Kieu T. M. Pham ◽  
Zhao-Rong Lun ◽  
Ziyin Li
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Bloodstream Form ◽  
Signaling Cascade ◽  
Insect Vector ◽  
Content Type ◽  
Procyclic Form ◽  
Life Cycle Stages ◽  
Evolutionarily Conserved

ABSTRACT The early divergent protozoan parasite Trypanosoma brucei alternates between the insect vector and the mammalian hosts during its life cycle and proliferates through binary cell fission. The cell cycle control system in T. brucei differs substantially from that in its mammalian hosts and possesses distinct mitosis-cytokinesis checkpoint controls between two life cycle stages, the procyclic form and the bloodstream form. T. brucei undergoes an unusual mode of cytokinesis, which is controlled by a novel signaling cascade consisting of evolutionarily conserved protein kinases and trypanosome-specific regulatory proteins in the procyclic form. However, given the distinct mitosis-cytokinesis checkpoints between the two forms, it is unclear whether the cytokinesis regulatory pathway discovered in the procyclic form also operates in a similar manner in the bloodstream form. Here, we showed that the three regulators of cytokinesis initiation, cytokinesis initiation factor 1 (CIF1), CIF2, and CIF3, are interdependent for subcellular localization but not for protein stability as in the procyclic form. Further, we demonstrated that KLIF, a regulator of cytokinesis completion in the procyclic form, plays limited roles in cytokinesis in the bloodstream form. Finally, we showed that the cleavage furrow-localizing protein FRW1 is required for cytokinesis initiation in the bloodstream form but is nonessential for cytokinesis in the procyclic form. Together, these results identify conserved and life cycle-specific functions of cytokinesis regulators, highlighting the distinction in the regulation of cytokinesis between different life cycle stages of T. brucei. IMPORTANCE The early divergent protozoan parasite Trypanosoma brucei is the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. This parasite has a complex life cycle by alternating between the insect vector and the mammalian hosts and proliferates by binary cell fission. The control of cell division in trypanosomes appears to be distinct from that in its human host and differs substantially between two life cycle stages, the procyclic (insect) form and the bloodstream form. Cytokinesis, the final step of binary cell fission, is regulated by a novel signaling cascade consisting of two evolutionarily conserved protein kinases and a cohort of trypanosome-specific regulators in the procyclic form, but whether this signaling pathway operates in a similar manner in the bloodstream form is unclear. In this report, we performed a functional analysis of multiple cytokinesis regulators and discovered their distinct functions and regulations in the bloodstream form.

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