Trypanosoma brucei vacuolar protein sorting 41 (VPS41) is required for intracellular iron utilization and maintenance of normal cellular morphology

Parasitology ◽  
2007 ◽  
Vol 134 (11) ◽  
pp. 1639-1647 ◽  
Author(s):  
S. LU ◽  
T. SUZUKI ◽  
N. IIZUKA ◽  
S. OHSHIMA ◽  
Y. YABU ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Iron Metabolism ◽  
Protein Sorting ◽  
Tsetse Fly ◽  
Cellular Morphology ◽  
Yeast Cells ◽  
Intracellular Iron ◽  
Procyclic Form ◽  
Vacuolar Protein ◽  
Iron Utilization

SUMMARYProcyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in ΔScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of ΔScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in ΔScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.

scholarly journals GPI-anchored Proteins and Free GPI Glycolipids of Procyclic Form Trypanosoma brucei Are Nonessential for Growth, Are Required for Colonization of the Tsetse Fly, and Are Not the Only Components of the Surface Coat

2006 ◽  
Vol 17 (12) ◽  
pp. 5265-5274 ◽  
Author(s):  
Maria Lucia Sampaio Güther ◽  
Sylvia Lee ◽  
Laurence Tetley ◽  
Alvaro Acosta-Serrano ◽  
Michael A.J. Ferguson
Keyword(s):  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Current Model ◽  
Apparent Molecular Weight ◽  
Tsetse Fly ◽  
Surface Coat ◽  
Procyclic Form ◽  
Cell Surface Biotinylation ◽  
A Cell

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine–containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine–containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI–anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.

scholarly journals Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (12) ◽  
pp. 6742-6754 ◽  
Author(s):  
P K Herman ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cell Fractionation ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Compartment ◽  
Dividing Cells ◽  
Vacuolar Protein

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

scholarly journals Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (12) ◽  
pp. 6742-6754
Author(s):  
P K Herman ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cell Fractionation ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Compartment ◽  
Dividing Cells ◽  
Vacuolar Protein

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

scholarly journals The Saccharomyces cerevisiae MVP1 gene interacts with VPS1 and is required for vacuolar protein sorting.

1995 ◽  
Vol 15 (3) ◽  
pp. 1671-1678 ◽  
Author(s):  
K Ekena ◽  
T H Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Protein Sorting ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Vacuolar Protein Sorting ◽  
Cellular Processes ◽  
Golgi Membranes ◽  
Multicopy Suppressors ◽  
Vacuolar Protein

The VPS1 gene of Saccharomyces cerevisiae encodes an 80-kDa GTPase that associates with Golgi membranes and is required for the sorting of proteins to the yeast vacuole. Vps1p is a member of a growing family of high-molecular-weight GTPases that are found in a number of organisms and are involved in a variety of cellular processes. Vps1p is most similar to mammalian dynamin and the Drosophila Shibire protein, both of which have been shown to play a role in an early step of endocytosis. To identify proteins that interact with Vps1p, a genetic screen was designed to isolate multicopy suppressors of dominant-negative vps1 mutations. One such suppressor, MVP1, that exhibits genetic interaction with VPS1 and is itself required for vacuolar protein sorting has been isolated. Overproduction of Mvp1p will suppress several dominant alleles of VPS1, and suppression is dependent on the presence of wild-type Vps1p. MVP1 encodes a 59-kDa hydrophilic protein, Mvp1p, which appears to colocalize with Vps1p in vps1d and vps27 delta yeast cells. We therefore propose that Mvp1p and Vps1p act in concert to promote membrane traffic to the vacuole.

scholarly journals Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins.

1989 ◽  
Vol 109 (1) ◽  
pp. 93-100 ◽  
Author(s):  
J H Rothman ◽  
C T Yamashiro ◽  
C K Raymond ◽  
P M Kane ◽  
T H Stevens
Keyword(s):  
Protein Sorting ◽  
Critical Role ◽  
Normal Function ◽  
Yeast Cells ◽  
Zymogen Activation ◽  
Animal Cells ◽  
Vacuolar Acidification ◽  
Vacuolar Protein ◽  
Vacuolar Membranes ◽  
Yeast Mutants

Organelle acidification plays a demonstrable role in intracellular protein processing, transport, and sorting in animal cells. We investigated the relationship between acidification and protein sorting in yeast by treating yeast cells with ammonium chloride and found that this lysosomotropic agent caused the mislocalization of a substantial fraction of the newly synthesized vacuolar (lysosomal) enzyme proteinase A (PrA) to the cell surface. We have also determined that a subset of the vpl mutants, which are deficient in sorting of vacuolar proteins (Rothman, J. H., and T. H. Stevens. 1986. Cell. 47:1041-1051; Rothman, J. H., I. Howald, and T. H. Stevens. EMBO [Eur. Mol. Biol. Organ.] J. In press), failed to accumulate the lysosomotropic fluorescent dye quinacrine within their vacuoles, mimicking the phenotype of wild-type cells treated with ammonium. The acidification defect of vpl3 and vpl6 mutants correlated with a marked deficiency in vacuolar ATPase activity, diminished levels of two immunoreactive subunits of the protontranslocating ATPase (H+-ATPase) in purified vacuolar membranes, and accumulation of the intracellular portion of PrA as the precursor species. Therefore, some of the VPL genes are required for the normal function of the yeast vacuolar H+-ATPase complex and may encode either subunits of the enzyme or components required for its assembly and targeting. Collectively, these findings implicate a critical role for acidification in vacuolar protein sorting and zymogen activation in yeast, and suggest that components of the yeast vacuolar acidification system may be identified by examining mutants defective in sorting of vacuolar proteins.

scholarly journals Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 683 ◽  
Author(s):  
Terry K. Smith ◽  
Frédéric Bringaud ◽  
Derek P. Nolan ◽  
Luisa M. Figueiredo
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Tsetse Fly ◽  
Metabolic Reprogramming ◽  
Mammalian Host ◽  
Insect Vector ◽  
Environmental Signals ◽  
Cellular Metabolic Activity

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

scholarly journals Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2962-2966 ◽  
Author(s):  
R Oria ◽  
L Sanchez ◽  
T Houston ◽  
MW Hentze ◽  
FY Liew ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Iron Metabolism ◽  
Transferrin Receptor ◽  
Regulatory Protein ◽  
Inhibitory Effect ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Intracellular Iron ◽  
Cellular Iron ◽  
Erythroleukemia Cells

Nitric oxide (NO) is known to increase the affinity of the intracellular iron-regulatory protein (IRP) for iron-response elements (IREs) in transferrin receptor and ferritin mRNAs, suggesting that it may act as a regulator of cellular iron metabolism. In this study, exogenous NO produced by adding the NO-generator S-nitroso-N-acetyl penicillamine gave a dose-dependent upregulation of transferrin receptor expression by K562 erythroleukemia cells and increased levels of transferrin receptor mRNA. NO did not affect the affinity of transferrin binding by the transferrin receptor. NO alone did not alter intracellular ferritin levels, but it did abrogate the inhibitory effect of the iron chelator desferrioxamine and potentiated the stimulatory effect of additional iron. NO also caused some increase in ferritin mRNA levels, which might mask any IRP-/IRE-mediated inhibitory effect of NO on ferritin translation. Although NO did not affect net iron uptake, it increased release of iron from K562 cells pulsed previously with 59Fe, and subcellular fractionation showed that it also increased the proportion of intracellular iron bound to ferritin. These findings provide direct evidence that NO can affect cellular iron metabolism and suggest that NO produced in vivo by activated bone marrow macrophages might affect erythropoiesis.

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