Isolation and characterization of subcellular membranes of Entamoeba invadens.

Vliet, H H van; Spies, F.; Linnemans, W. A. M.; Klepke, A; Kamp, Johannes; Deenen, L L van

doi:10.1083/jcb.71.2.357

Cited by 22 publications

(16 citation statements)

References 40 publications

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“…In addition to 5'-mononucleotidase, Na+/K+-stimulated Mg-ATPase is another widely used surface membrane marker enzyme. In E. invadens a Mg-ATPase of low specific activity was found though not exclusively in the plasma membrane in subcellular fractions isolated by both McLaughlin & Meerovitch (148) and Van Vliet et al (240), the former authors noting slight stimulation in the presence of Na+ and K+. Unfortunately, in neither case was the specificity of the ATPase established using ouabain, a specific inhibitor of such ATPases.…”

Section: Surface Membrane Isolation and Marker Enzymesmentioning

confidence: 92%

“…A number of investigators (6,148,219,240) have since purportedly isolated surface membrane from both E. invadens and E. histolytica, attempting to use marker enzymes as indices of purification. In the study of McLaughlin & Meerovitch (148), the paucity of reliable endogenous markers for use in characterizing E. invadens subcellular fractions was revealed, including in agreement with a subsequent study of E. invadens (240), the absence of 5'-mononucleotidase, a widely used enzyme marker for plasma membrane (73). In E. histolytica, Serrano et al (2 19) claimed the presence of a distinct 5'-mononucleotidase, assayed at pH 7.5, which was found exclusively in the presumptive plasma membrane fraction.…”

Section: Surface Membrane Isolation and Marker Enzymesmentioning

confidence: 99%

“…It can be concluded that, to date, it has not been possible to identify any enzyme activity associated exclusively with the surface membrane of Entamoeba species. There is some evidence for a distinct surface membrane acid phosphatase in E. invadens (148,240) displaying a preference for p-nitrophenylphosphate rather than /3-glycerophosphate. Serrano et al (2 19) claimed that acid phosphatase (p-nitrophenylphosphate) was almost exclusively associated with the surface membrane of E. histolytica.…”

Section: Surface Membrane Isolation and Marker Enzymesmentioning

confidence: 99%

See 2 more Smart Citations

The Biochemistry and Functional Morphology of the Entamoeba1

McLaughlin

Aley

1985

The Journal of Protozoology

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Section: Surface Membrane Isolation and Marker Enzymesmentioning

confidence: 92%

Section: Surface Membrane Isolation and Marker Enzymesmentioning

confidence: 99%

Section: Surface Membrane Isolation and Marker Enzymesmentioning

confidence: 99%

See 1 more Smart Citation

The Biochemistry and Functional Morphology of the Entamoeba1

McLaughlin

Aley

1985

The Journal of Protozoology

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“…The Ca 2ϩ affinity of C2 domains has been observed to increase when the cognate target membrane lipid composition is recreated for in vitro determinations (11). Unfortunately, attempts to define the exact lipid composition of the Entamoeba plasma membrane, the target of EhC2A, have been limited (1,43).…”

Section: Discussionmentioning

confidence: 99%

Phospholipid-Binding Protein EhC2A Mediates Calcium-Dependent Translocation of Transcription Factor URE3-BP to the Plasma Membrane of Entamoeba histolytica

Moreno

Gilchrist²,

Petri

2010

Eukaryot Cell

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The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca 2؉ -inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. The parasite Entamoeba histolytica is the causative agent of amebiasis, estimated to be the second leading protozoan cause of morbidity and mortality in humans worldwide (2). The molecular mechanisms that regulate the outcome of infection have yet to be elucidated; however, we hypothesize that transcriptional regulation may play an essential role in determining such outcomes.The calcium ion (Ca 2ϩ ) is a universal regulator of many cellular processes in eukaryotic cells, including cell signaling events, gene expression, and cell death. In Entamoeba, Ca 2ϩ has been implicated in many aspects of the parasite's pathophysiology, including cell motility (35), adhesion to fibronectin (6), cytolytic activity (36), transcriptional regulation (16-18), and growth and developmental stage transition (25, 26). In eukaryotes, these processes are generally mediated by proteins possessing Ca 2ϩ -binding motifs, such as EF-hands, endonexin folds, and C2 domains, which, in response to changes in intracellular Ca 2ϩ levels, are responsible for the transduction of signaling events, several of which have been identified in Entamoeba (for a review, see reference 4).The upstream regulatory element 3-binding protein (URE3-BP) is an E. histolytica Ca 2ϩ -regulated transcription factor first identified for its roles in the control of the expression of ferredoxin (fdx1) and the heavy subunit of the galactose/N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin (hgl5). The protein was isolated by a yeast one-hybrid screen using the cognate URE3 DNA motif (17), though the amino acid sequence of URE3-BP revealed no DNA-binding motifs. The protein did, however, possess two canonical and one noncanonical EF-hand motifs. URE3-BP localized to both the nucleus and the cytoplasm of trophozoites and was also found to be enriched in the plasma membrane. Ca 2ϩ induced the release of binding of URE3-BP from the URE3 motif in vitro and in vivo (18). Mutation of the second EF-hand motif at residues critical for Ca 2ϩ coordination abrogated this effect and allowed for the genome-wide identification of targets of URE3-BP in parasites overexpressing the mutant protein (16).In this study, we explored the reason for the unusual plasma membrane localization of the URE3-BP transcription factor. We discovered a novel URE3-BP binding partner, EhC2A, containing an N-terminal Ca 2ϩ -binding C2 domain and a proline-rich C-terminal region. C2 domains are ϳ120-residue motifs that confer phospholipid-binding activity on the majority of proteins which possess them (reviewed in references 9, 22, and 37). EhC2A coimmunoprecipitated with URE3-BP in a Ca 2ϩ -dependent manner and ...

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“…Since Entamoeba membranes contain cholesterol (64), it is conceivable that raft-like domains exist in the plasma membrane of this organism. Therefore, we have conducted a study to identify and characterize raft-like domains in this pathogen.…”

mentioning

confidence: 99%

Involvement of Raft-Like Plasma Membrane Domains of Entamoeba histolytica in Pinocytosis and Adhesion

Laughlin¹,

McGugan

Powell

et al. 2004

Infect Immun

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Lipid rafts are highly ordered, cholesterol-rich, and detergent-resistant microdomains found in the plasma membrane of many eukaryotic cells. These domains play important roles in endocytosis, secretion, and adhesion in a variety of cell types. The parasitic protozoan Entamoeba histolytica, the causative agent of amoebic dysentery, was determined to have raft-like plasma membrane domains by use of fluorescent lipid analogs that specifically partition into raft and nonraft regions of the membrane. Disruption of raft-like membrane domains in Entamoeba with the cholesterol-binding agents filipin and methyl-␤-cyclodextrin resulted in the inhibition of several important virulence functions, fluid-phase pinocytosis, and adhesion to host cell monolayers. However, disruption of raft-like domains did not inhibit constitutive secretion of cysteine proteases, another important virulence function of Entamoeba. Flotation of the cold Triton X-100-insoluble portion of membranes on sucrose gradients revealed that the heavy, intermediate, and light subunits of the galactose-N-acetylgalactosamine-inhibitible lectin, an important cell surface adhesion molecule of Entamoeba, were enriched in cholesterol-rich (raft-like) fractions, whereas EhCP5, another cell surface molecule, was not enriched in these fractions. The subunits of the lectin were also observed in high-density, actin-rich fractions of the sucrose gradient. Together, these data suggest that pinocytosis and adhesion are raft-dependent functions in this pathogen. This is the first report describing the existence and physiological relevance of raft-like membrane domains in E. histolytica.Recent evidence suggests that plasma membrane lipids are nonhomogeneously distributed and that microdomains with specialized functions exist in the membrane. One such domain, a lipid raft, is a highly ordered, less-fluid, and tightly packaged membrane domain enriched in cholesterol (or other sterols), glycosphingolipids, and phospholipids with a higher degree of saturated fatty acyl chains than those of the rest of the membrane (reviewed in references 37 and 54). These domains are also detergent insoluble and are thus often referred to as detergent-resistant membranes (DRMs). The presence of microdomains in membranes allows for the inclusion or exclusion of membrane proteins based on their attachment to the membrane via lipid anchors or specific protein-lipid interactions. For example, proteins that are modified with a hydrophobic attachment, such as a glycosylphosphatidylinositol (GPI) anchor, or double acylation or transmembrane proteins with the capacity to interact with cholesterol are often found in lipid microdomains.The physiological role of lipid rafts has been the subject of numerous recent studies, and it has recently become clear that these membrane regions play an important role in a variety of cellular functions, including polarization, signal transduction, endocytosis, secretion, and cell-cell and cell-pathogen adhesion (17,18,21,34,36,44). A range of cell surface receptors m...

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Isolation and characterization of subcellular membranes of Entamoeba invadens.

Cited by 22 publications

References 40 publications

The Biochemistry and Functional Morphology of the Entamoeba1

The Biochemistry and Functional Morphology of the Entamoeba1

Phospholipid-Binding Protein EhC2A Mediates Calcium-Dependent Translocation of Transcription Factor URE3-BP to the Plasma Membrane of Entamoeba histolytica

Involvement of Raft-Like Plasma Membrane Domains of Entamoeba histolytica in Pinocytosis and Adhesion

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