scholarly journals Functional Complementation Analyses Reveal that the Single PRAT Family Protein of Trypanosoma brucei Is a Divergent Homolog of Tim17 in Saccharomyces cerevisiae

2015 ◽  
Vol 14 (3) ◽  
pp. 286-296 ◽  
Author(s):  
Ebony Weems ◽  
Ujjal K. Singha ◽  
VaNae Hamilton ◽  
Joseph T. Smith ◽  
Karin Waegemann ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trypanosoma Brucei ◽  
Mitochondrial Proteins ◽  
Growth Defect ◽  
Heterologous Proteins ◽  
Homologous Proteins ◽  
Content Type ◽  
Mitochondrial Inner Membrane ◽  
Family Protein ◽  
Higher Eukaryotes

ABSTRACT Trypanosoma brucei , a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequence and amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins, ScTim23, ScTim17, and ScTim22, are found in Saccharomyces cerevisiae and higher eukaryotes. Here, we show that TbTim17 cannot rescue Tim17, Tim23, or Tim22 mutants of S. cerevisiae . We expressed S. cerevisiae Tim23, Tim17, and Tim22 in T. brucei . These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that although ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complex similar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensitive soluble protein in the T. brucei mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in T. brucei was partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expression of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findings for cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part restored by the expression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggest that TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 could not be sorted properly in the T. brucei mitochondrion and thus failed to complement the function of TbTim17.

scholarly journals The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

2015 ◽  
Vol 14 (3) ◽  
pp. 297-310 ◽  
Author(s):  
Anna Gnipová ◽  
Karolína Šubrtová ◽  
Brian Panicucci ◽  
Anton Horváth ◽  
Julius Lukeš ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Trypanosoma Brucei ◽  
Atp Synthesis ◽  
Model Organisms ◽  
Growth Defect ◽  
Respiratory Chain Complexes ◽  
Content Type ◽  
Chain Complexes ◽  
Parasitic Protist ◽  
Veterinary Importance

ABSTRACT The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei , a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote.

scholarly journals Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in Trypanosoma brucei

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Joseph T. Smith ◽  
Ujjal K. Singha ◽  
Smita Misra ◽  
Minu Chaudhuri
Keyword(s):  
Membrane Proteins ◽  
Trypanosoma Brucei ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Content Type ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Intermembrane Space ◽  
The Stability ◽  
Tim Proteins

ABSTRACT The small Tim proteins belong to a group of mitochondrial intermembrane space chaperones that aid in the import of mitochondrial inner membrane proteins with internal targeting signals. Trypanosoma brucei , the protozoan parasite that causes African trypanosomiasis, possesses multiple small Tim proteins that include homologues of T. brucei Tim9 (TbTim9) and Tim10 (TbTim10) and a unique small Tim that shares homology with both Tim8 and Tim13 (TbTim8/13). Here, we found that these three small TbTims are expressed as soluble mitochondrial intermembrane space proteins. Coimmunoprecipitation and mass spectrometry analysis showed that the small TbTims stably associated with each other and with TbTim17, the major component of the mitochondrial inner membrane translocase in T. brucei . Yeast two-hybrid analysis indicated direct interactions among the small TbTims; however, their interaction patterns appeared to be different from those of their counterparts in yeast and humans. Knockdown of the small TbTims reduced cell growth and decreased the steady-state level of TbTim17 and T. brucei ADP/ATP carrier (TbAAC), two polytopic mitochondrial inner membrane proteins. Knockdown of small TbTims also reduced the matured complexes of TbTim17 in mitochondria. Depletion of any of the small TbTims reduced TbTim17 import moderately but greatly hampered the stability of the TbTim17 complexes in T. brucei . Altogether, our results revealed that TbTim9, TbTim10, and TbTim8/13 interact with each other, associate with TbTim17, and play a crucial role in the integrity and maintenance of the levels of TbTim17 complexes. IMPORTANCE Trypanosoma brucei is the causative agent of African sleeping sickness. The parasite’s mitochondrion represents a useful source for potential chemotherapeutic targets. Similarly to yeast and humans, mitochondrial functions depend on the import of proteins that are encoded in the nucleus and made in the cytosol. Even though the machinery involved in this mitochondrial protein import process is becoming clearer in T. brucei , a comprehensive picture of protein complex composition and function is still lacking. In this study, we characterized three T. brucei small Tim proteins, TbTim9, TbTim10, and TbTim8/13. Although the parasite does not have the classical TIM22 complex that imports mitochondrial inner membrane proteins containing internal targeting signals in yeast or humans, we found that these small TbTims associate with TbTim17, the major subunit of the TbTIM complex in T. brucei , and play an essential role in the stability of the TbTim17 complexes. Therefore, these divergent proteins are critical for mitochondrial protein biogenesis in T. brucei .

scholarly journals A Genomewide Screen for Tolerance to Cationic Drugs Reveals Genes Important for Potassium Homeostasis in Saccharomyces cerevisiae

2011 ◽  
Vol 10 (9) ◽  
pp. 1241-1250 ◽  
Author(s):  
Lina Barreto ◽  
David Canadell ◽  
Silvia Petrezsélyová ◽  
Clara Navarrete ◽  
Lydie Marešová ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Potassium Concentration ◽  
Growth Defect ◽  
Electrochemical Gradient ◽  
Biochemical Tests ◽  
Content Type ◽  
Potassium Homeostasis ◽  
Moderate Growth ◽  
Number Of Genes ◽  
Mutant Strains

ABSTRACTPotassium homeostasis is crucial for living cells. In the yeastSaccharomyces cerevisiae, the uptake of potassium is driven by the electrochemical gradient generated by the Pma1 H+-ATPase, and this process represents a major consumer of the gradient. We considered that any mutation resulting in an alteration of the electrochemical gradient could give rise to anomalous sensitivity to any cationic drug independently of its toxicity mechanism. Here, we describe a genomewide screen for mutants that present altered tolerance to hygromycin B, spermine, and tetramethylammonium. Two hundred twenty-six mutant strains displayed altered tolerance to all three drugs (202 hypersensitive and 24 hypertolerant), and more than 50% presented a strong or moderate growth defect at a limiting potassium concentration (1 mM). Functional groups such as protein kinases and phosphatases, intracellular trafficking, transcription, or cell cycle and DNA processing were enriched. Essentially, our screen has identified a substantial number of genes that were not previously described to play a direct or indirect role in potassium homeostasis. A subset of 27 representative mutants were selected and subjected to diverse biochemical tests that, in some cases, allowed us to postulate the basis for the observed phenotypes.

scholarly journals The Ancient Small GTPase Rab21 Functions in Intermediate Endocytic Steps in Trypanosomes

2013 ◽  
Vol 13 (2) ◽  
pp. 304-319 ◽  
Author(s):  
Moazzam Ali ◽  
Ka Fai Leung ◽  
Mark C. Field
Keyword(s):  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Cellular Proliferation ◽  
Small Gtpase ◽  
Eukaryotic Cells ◽  
Content Type ◽  
Endosomal Sorting ◽  
Intimate Connection ◽  
Late Endosomes ◽  
Higher Eukaryotes

ABSTRACTEndocytosis is an essential process in nearly all eukaryotic cells, including the African trypanosomeTrypanosoma brucei. Endocytosis in these organisms is exclusively clathrin mediated, although several lineage-specific features indicate that precise mechanisms are distinct from those of higher eukaryotes.T. bruceiRab21 is a member of an ancient, pan-eukaryotic, endocytic Rab clade that is retained by trypanosomes. We show thatT. bruceiRab21 (TbRab21) localizes to endosomes, partially colocalizing with TbRab5A, TbRab28, and TbVps23, the latter two being present at late endosomes. TbRab21 expression is essential for cellular proliferation, and its suppression results in a partial block in traffic to the lysosome. RNA interference (RNAi)-mediated knockdown of TbRab21 had no effect on TbRab5A expression or location but did result in decreased intransexpression of ESCRT (trypanosome endosomal sorting complex required for transport) components and TbRab28, while knockdown of ESCRT subunit TbVps23 resulted in decreased TbRab21 expression. These data suggest that TbRab21 acts downstream of TbRab5A and functions in intimate connection with the trypanosome ESCRT system.

scholarly journals Pyrophosphate Stimulates the Phosphate-Sodium Symporter ofTrypanosoma bruceiAcidocalcisomes andSaccharomyces cerevisiaeVacuoles

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Evgeniy Potapenko ◽  
Ciro D. Cordeiro ◽  
Guozhong Huang ◽  
Roberto Docampo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Potential ◽  
Trypanosoma Brucei ◽  
Wild Type ◽  
Inorganic Pyrophosphate ◽  
Content Type ◽  
Yeast Strains ◽  
Outward Currents ◽  
Sodium Dependent

ABSTRACTInorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPiand other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Piconductance inXenopusoocytes expressing aSaccharomyces cerevisiaeorTrypanosoma bruceiNa+/Pisymporter. PPiis not taken up byXenopusoocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPigenerated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type orpho91Δ yeast strains expressingTbPHO91but not from thepho91Δstrains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing PifromS. cerevisiaevacuoles andT. bruceiacidocalcisomes.IMPORTANCEAcidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities withS. cerevisiaevacuoles in their structure and composition. Both organelles share a Na+/Pisymporter involved in Pirelease to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in eitherXenopusoocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.

scholarly journals Lsm12 Mediates Deubiquitination of DNA Polymerase η To Help Saccharomyces cerevisiae Resist Oxidative Stress

2018 ◽  
Vol 85 (1) ◽  
Author(s):  
Rui Yao ◽  
Liujia Shi ◽  
Chengjin Wu ◽  
Weihua Qiao ◽  
Liming Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Cell Growth ◽  
Dna Polymerase ◽  
Genome Stability ◽  
Fold Increase ◽  
Growth Defect ◽  
Physical Interaction ◽  
Content Type

ABSTRACT In Saccharomyces cerevisiae, the Y family DNA polymerase η (Polη) regulates genome stability in response to different forms of environmental stress by translesion DNA synthesis. To elucidate the role of Polη in oxidative stress-induced DNA damage, we deleted or overexpressed the corresponding gene RAD30 and used transcriptome analysis to screen the potential genes associated with RAD30 to respond to DNA damage. Under 2 mM H2O2 treatment, the deletion of RAD30 resulted in a 2.2-fold decrease in survival and a 2.8-fold increase in DNA damage, whereas overexpression of RAD30 increased survival and decreased DNA damage by 1.2- and 1.4-fold, respectively, compared with the wild-type strain. Transcriptome and phenotypic analyses identified Lsm12 as a main factor involved in oxidative stress-induced DNA damage. Deleting LSM12 caused growth defects, while its overexpression enhanced cell growth under 2 mM H2O2 treatment. This effect was due to the physical interaction of Lsm12 with the UBZ domain of Polη to enhance Polη deubiquitination through Ubp3 and consequently promote Polη recruitment. Overall, these findings demonstrate that Lsm12 is a novel regulator mediating Polη deubiquitination to promote its recruitment under oxidative stress. Furthermore, this study provides a potential strategy to maintain the genome stability of industrial strains during fermentation. IMPORTANCE Polη was shown to be critical for cell growth in the yeast Saccharomyces cerevisiae, and deletion of its corresponding gene RAD30 caused a severe growth defect under exposure to oxidative stress with 2 mM H2O2. Furthermore, we found that Lsm12 physically interacts with Polη and promotes Polη deubiquitination and recruitment. Overall, these findings indicate Lsm12 is a novel regulator mediating Polη deubiquitination that regulates its recruitment in response to DNA damage induced by oxidative stress.

scholarly journals Homologue replacement in the import motor of the mitochondrial inner membrane of trypanosomes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Corinne von Känel ◽  
Sergio A Muñoz-Gómez ◽  
Silke Oeljeklaus ◽  
Christoph Wenger ◽  
Bettina Warscheid ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Driving Force ◽  
Protein Import ◽  
Normal Growth ◽  
Mitochondrial Proteins ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Bona Fide ◽  
J Domain ◽  
J Protein

Many mitochondrial proteins contain N-terminal presequences that direct them to the organelle. The main driving force for their translocation across the inner membrane is provided by the presequence translocase-associated motor (PAM) which contains the J-protein Pam18. Here, we show that in the PAM of Trypanosoma brucei the function of Pam18 has been replaced by the non-orthologous euglenozoan-specific J-protein TbPam27. TbPam27 is specifically required for the import of mitochondrial presequence-containing but not for carrier proteins. Similar to yeast Pam18, TbPam27 requires an intact J-domain to function. Surprisingly, T. brucei still contains a bona fide Pam18 orthologue that, while essential for normal growth, is not involved in protein import. Thus, during evolution of kinetoplastids, Pam18 has been replaced by TbPam27. We propose that this replacement is linked to the transition from two ancestral and functionally distinct TIM complexes, found in most eukaryotes, to the single bifunctional TIM complex present in trypanosomes.

scholarly journals The Cross Talk between TbTim50 and PIP39, Two Aspartate-Based Protein Phosphatases, Maintains Cellular Homeostasis in Trypanosoma brucei

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Anuj Tripathi ◽  
Ujjal K. Singha ◽  
Victor Paromov ◽  
Salisha Hill ◽  
Siddharth Pratap ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Cross Talk ◽  
Developmental Regulation ◽  
Protein Phosphatases ◽  
Infectious Agent ◽  
Cellular Stress Response ◽  
Content Type ◽  
Mitochondrial Inner Membrane ◽  
Inner Membrane Protein ◽  
Metabolic Activities

Trypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.

scholarly journals Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

2004 ◽  
Vol 72 (10) ◽  
pp. 5983-5992 ◽  
Author(s):  
Jessica A. Sexton ◽  
Jennifer L. Miller ◽  
Aki Yoneda ◽  
Thomas E. Kehl-Fie ◽  
Joseph P. Vogel
Keyword(s):  
Cell Surface ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Wide Distribution ◽  
Host Cells ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Homologous Proteins ◽  
Type Iv ◽  
Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

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