Immunolocalization of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase to the glycosome

Shih, Sarah; Stenberg, Paula E.; Ullman, Buddy

doi:10.1016/s0166-6851(98)00007-3

Cited by 9 publications

(5 citation statements)

References 13 publications

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“…Interestingly, whereas trypanosomatid IMPDH E shares many critical residues with the human enzyme (identical residues shown in black), IMDPH P sequences are be more similar to the prokaryotic enzymes (identical residues in red). Both IMPDH E and IMPDH P harbor a C-terminal glycosomal targeting motif as previously shown for other enzymes in the trypanosomatid purine salvage pathway [370,371].…”

Section: Impdh Genessupporting

confidence: 55%

The early evolution of meiotic genes

Malik¹

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Thesis Supervisor: Associate Professor John M. Logsdon Jr.Meiosis is a hallmark of eukaryotes. However, to demonstrate that homologous sexual processes are pan-eukaryotic, it is necessary to compare the molecular machinery of meiotic recombination across diverse eukaryotic groups, especially protists. The homologs of 34 meiotic genes encoding components of the meiotic recombination machinery, sister chromatid cohesion and synaptonemal complexes were identified in the genomes of diverse eukaryotes and verified by phylogenetic analyses. Twelve of these genes function only in meiosis in model organisms Hop1, Hop2, Mnd1, Dmc1, Msh4, Msh5, Mer3, Zip1, Zip4 and Rec8). Homologs of meiosis-specific genes were identified in several organisms previously considered to be asexual, indicating that they are derived from sexual lineages, and may have as-yet-unobserved meiosis. The putatively early-diverged protists Trichomonas and Giardia have orthologs of eight and six of the meiosis-specific genes, respectively. Using degenerate PCR, additional meiosis-specific genes were identified in Naegleria, Acanthamoeba, Amphidinium and other protists. All of the meiotic genes are conserved in animals, fungi and plants; most of the meiotic genes (including the meiosis-specific genes) are also broadly conserved among protist lineages. These data indicate that the molecular machinery for meiotic recombination evolved once, early during eukaryotic evolution, prior to the divergence of major eukaryotic lineages. Thus, the eukaryotic cenancestor likely had each component of this "meiosis detection toolkit". Many of the 34 meiotic genes -including ten meiosis-specific genes -evolved by gene duplications early during eukaryotic evolution, revealing that gene duplication has been a pervasive evolutionary force in the evolution of the meiotic machinery. Abstract Approved: ____________________________________ Thesis Supervisor ____________________________________ ii To my grandmothers, Bano-bai Mazher Master binte Imran-bhai Shamsi and the late Zainab-bai Gulamali Malik binte Noman-bhai Rangwala, and to my mother, Nafisa Hatim Malik binte Mazher-bhai Master. iii ACKNOWLEDGMENTS I would like to express my gratitude towards the many people who contributed towards my growth as a scientist while I have pursued this degree, from Emory University to the University of Iowa. First and foremost, I would like to thank my thesis advisor, John Logsdon, for his time, advice, many discussions, and for giving me the opportunity to work with him on the projects culminating in this thesis. I also feel fortunate that John has sent me to present my work at a conference almost every year, and really helped me to network with other scientists. I am grateful to my other thesis advisory committee members, Bryant McAllister, Bob Malone, Debashish Bhattacharya and Chris Brochu, for their time, constructive criticism and helpful advice. I am indebted to Mark Farmer and David Patterson for illustrative discussions about protists. I am thankful to Chris Beck, Sonia Altiz...

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Section: Impdh Genessupporting

confidence: 55%

The early evolution of meiotic genes

Malik¹

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“…On the other hand binding of glycosomal enzymes to polyP could be explained by the presence of binding domains to polyP. Several glycosomal enzymes found in our proteome analyses, like hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) (Shih et al , 1998) and pyruvate phosphate dikinase (PPDK) (Bringaud et al , 1998, Maldonado & Fairlamb, 2001, Shih et al , 1998) produce or utilize PPi. Phosphofructokinase (PFK) is an ATP-dependent enzyme but shows a high degree of sequence and structural similarity with PPi-dependent enzymes (Rodriguez E., 2009, Michels et al , 1997, McNae et al , 2009).…”

Section: Discussionmentioning

confidence: 91%

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids

Negreiros

Lander

Huang

et al. 2018

Molecular Microbiology

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Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

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“…They obtain their purines by taking up nucleobases and nucleosides from their environment (12,38) and interconvert them using various purine phosphoribosyltransferases. Several of the purine salvage enzymes have been shown to be localized in the glycosome (15,72,91), implying that the glycosomal membrane must contain appropriate transporters. Also, the mitochondria must contain such transporters, since nucleotides required for DNA replication and RNA synthesis need to be imported from the cytosol.…”

Section: Discussionmentioning

confidence: 99%

Characterization and Developmentally Regulated Localization of the Mitochondrial Carrier Protein Homologue MCP6 from Trypanosoma brucei

et al. 2006

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The order Kinetoplastida includes among its members a number of important human and veterinary pathogens, such as the African trypanosome Trypanosoma brucei (83). This parasite has a complex life cycle that alternates between the bloodstream form found in the blood and tissue fluids of mammals and the procyclic (insect) form in the midgut of the tsetse fly (46, 83). T. brucei undergoes a series of metabolic and morphological changes in order to adapt to these distinct host environments (45, 46). Bloodstream-form T. brucei metabolizes glucose to pyruvate and glycerol, obtaining ATP by substrate-level phosphorylation during glycolysis (48, 55). The mitochondria of this life form are highly reduced and lack key enzymes and components of the tricarboxylic acid cycle; their role in energy metabolism is probably restricted to the reoxidation of glycerol-3 phosphate by the mitochondrial alternative oxidase (20,48). This is in contrast to the procyclic form, which metabolizes amino acids and has a well-developed mitochondrion in which ATP is generated by a combination of substratelevel and oxidative phosphorylation (16,48,73,82).In both life forms of T. brucei, most of the glycolytic enzymes are found within a peroxisome-like organelle called the glycosome (14,48,60,61). This unique compartmentation of glycolytic enzymes has been shown to be essential for trypanosome survival (5,11,22,25,51). It has been proposed that the glycosomal membrane forms a barrier to ATP and ADP, insulating the enzymes of the glycosomal matrix from the ADP/ ATP ratio in the cytosol (5).So far, no glycosomal or mitochondrial metabolite carriers have been identified for T. brucei, although they most probably play a key role in the regulation of energy metabolism. The characterization of these carriers should make it possible to build more-accurate biochemical and mathematical models of trypanosome energy metabolism (5).The mitochondrial carrier family (MCF) was initially defined as a group of proteins that are located in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates (3,32,35,(57)(58)(59)86). Recently, several structurally and functionally related carrier proteins were also found in the membranes of peroxisomes (52,81,88,90). We therefore wondered whether such proteins might also be found in kinetoplastid glycosomes. The recently completed genome sequence of T. brucei (8) contains about 29 genes encoding different proteins of the mitochondrial carrier family. In this paper, we describe the characterization of MCP6, a novel mitochondrial carrier protein homologue from T. brucei. MATERIALS AND METHODSCulture and transfection. Trypanosoma brucei cell lines were cultured in either HMI-9 medium for bloodstream cell lines (29) or MEM-PROS medium for procyclic cell lines (56), supplemented with 10% (vol/vol) fetal calf serum (Sigma-Aldrich). Cells were transfected as described previously (9). In all experiments, "wild type" refers to bloodstream or procyclic T. brucei strain

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Immunolocalization of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase to the glycosome

Cited by 9 publications

References 13 publications

The early evolution of meiotic genes

The early evolution of meiotic genes

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids

Characterization and Developmentally Regulated Localization of the Mitochondrial Carrier Protein Homologue MCP6 from Trypanosoma brucei

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