scholarly journals Overexpression of a Mutant Form of EhRabA, a Unique Rab GTPase of Entamoeba histolytica, Alters Endoplasmic Reticulum Morphology and Localization of the Gal/GalNAc Adherence Lectin

2009 ◽  
Vol 8 (7) ◽  
pp. 1014-1026 ◽  
Author(s):  
B. H. Welter ◽  
L. A. Temesvari
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Entamoeba Histolytica ◽  
Vesicle Trafficking ◽  
Brefeldin A ◽  
Surface Proteins ◽  
Secretory Proteins ◽  
Rab Gtpase ◽  
Mutant Form ◽  
Rab Gtpases

ABSTRACT Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/N-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica.

scholarly journals Cell surface proteins of Entamoeba histolytica

1991 ◽  
Vol 7 (7) ◽  
pp. 173-176 ◽  
Author(s):  
B.J. Mann ◽  
W.A. Petri
Keyword(s):  
Cell Surface ◽  
Entamoeba Histolytica ◽  
Surface Proteins ◽  
Cell Surface Proteins

scholarly journals Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

1999 ◽  
Vol 10 (4) ◽  
pp. 1043-1059 ◽  
Author(s):  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Translocation ◽  
Sequence Similarity ◽  
Surface Proteins ◽  
Golgi Transport ◽  
Er Membrane

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes inSaccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting thatLAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δcells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

scholarly journals The discrepancy between presenilin subcellular localization and γ-secretase processing of amyloid precursor protein

2001 ◽  
Vol 154 (4) ◽  
pp. 731-740 ◽  
Author(s):  
Philippe Cupers ◽  
Mustapha Bentahir ◽  
Katleen Craessaerts ◽  
Isabelle Orlans ◽  
Hugo Vanderstichele ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Amyloid Precursor Protein ◽  
Primary Cultures ◽  
Brefeldin A ◽  
Precursor Protein ◽  
Subcellular Compartment ◽  
Marker Proteins ◽  
Intermediate Compartment ◽  
The Relationship

We investigated the relationship between PS1 and γ-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent γ-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no γ-secretase processing was observed when holo-APP or APP-C99, a direct substrate for γ-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent γ-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that γ-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the γ-cleavage of APP-C99. In agreement, we found that intracellular γ-secretase processing of APP-C99-KK both at the γ40 and the γ42 site could be restored partially after brefeldin A treatment. Our data confirm the “spatial paradox” and raise several questions regarding the PS1 is γ-secretase hypothesis.

scholarly journals Synthesis and trafficking of prion proteins in cultured cells.

1992 ◽  
Vol 3 (8) ◽  
pp. 851-863 ◽  
Author(s):  
A Taraboulos ◽  
A J Raeber ◽  
D R Borchelt ◽  
D Serban ◽  
S B Prusiner
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Cultured Cells ◽  
Prion Proteins ◽  
Brefeldin A ◽  
Endocytic Pathway ◽  
Chromosomal Gene ◽  
Golgi Stack ◽  
Mouse Neuroblastoma ◽  
N Terminus

Scrapie prions are composed largely, if not entirely, of the scrapie prion protein (PrPSc) that is encoded by a chromosomal gene. Scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells synthesize PrPSc from the normal PrP isoform (PrPC) or a precursor through a posttranslational process. In pulse-chase radiolabeling experiments, we found that presence of brefeldin A (BFA) during both the pulse and the chase periods prevented the synthesis of PrPSc. Removal of BFA after the chase permitted synthesis of PrPSc to resume. BFA also blocked the export of nascent PrPC to the cell surface but did not alter the distribution of intracellular deposits of PrPSc. Under the same conditions, BFA caused the redistribution of the Golgi marker MG160 into the endoplasmic reticulum (ER). Using monensin as an inhibitor of mid-Golgi glycosylation, we determined that PrP traverses the mid-Golgi stack before acquiring protease resistance. About 1 h after the formation of PrPSc, its N-terminus was removed by a proteolytic process that was inhibited by ammonium chloride, chloroquine, and monensin, arguing that this is a lysosomal event. These results suggest that the ER is not competent for the synthesis of PrPSc and that the synthesis of PrPSc occurs during the transit of PrP between the mid-Golgi stack and lysosomes. Presumably, the endocytic pathway features in the synthesis of PrPSc.

scholarly journals Brefeldin A inhibits the endocytosis of plasma-membrane-associated heparan sulphate proteoglycans of cultured rat ovarian granulosa cells

1995 ◽  
Vol 310 (1) ◽  
pp. 271-278 ◽  
Author(s):  
L Uhlin-Hansen ◽  
M Yanagishita
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Granulosa Cells ◽  
Protein Transport ◽  
Heparan Sulphate ◽  
Brefeldin A ◽  
Inhibitory Effect ◽  
Surface Proteins ◽  
Gel Chromatography ◽  
Ovarian Granulosa Cells

Rat ovarian granulosa cells were labelled with [35S]sulphate for 0.5-20 h and chased in the presence or absence of 1-2 micrograms/ml of brefeldin A (BFA) for up to 21 h. Heparan [35S]sulphate (HS) proteoglycans from the culture medium, plasma membrane and intracellular fractions were then analysed by gel chromatography. In the absence of BFA, about 85% of the plasma membrane-associated HS proteoglycans were endocytosed and subsequently degraded intracellularly. Recirculation of the HS proteoglycans between the intracellular pool and the cell surface was not observed. Exposing the cells to BFA for less than 1 h did not influence the turnover of the HS proteoglycans, whereas the effect of the drug on the Golgi functions reached a maximum in approx. 10 min. When the cells were treated with BFA for more than 1-2 h, the rate of endocytosis of HS proteoglycans was reduced to about 50% of the control. The delivery of endocytosed HS proteoglycans to lysosomes were not affected by the drug. Cycloheximide also reduced the endocytosis of HS proteoglycans, but not as much as BFA, indicating that the inhibitory effect of BFA can be only partly accounted for by a block of protein transport from the endoplasmic reticulum to the plasma membrane. In contrast with the endocytosis of HS proteoglycans, neither that of 125I-transferrin, known to be mediated by clathrin-coated vesicles, nor that of 125I-ricin, a marker molecule for bulk endocytosis, was affected by BFA. The half-life of 125I-transferrin and 125I-ricin in the plasma membrane was about 10 and 25 min respectively compared with about 5 h for the HS proteoglycans. Altogether, these results indicate that the endocytosis of plasma-membrane-associated HS proteoglycans is mediated by different mechanisms than the endocytosis of most other cell-surface proteins. Further, the mechanisms involved in the endocytosis of HS proteoglycans are sensitive to BFA.

Regulation of the trafficking and the function of the metalloprotease ADAM10 by tetraspanins

2017 ◽  
Vol 45 (4) ◽  
pp. 937-944 ◽  
Author(s):  
Julien Saint-Pol ◽  
Etienne Eschenbrenner ◽  
Emmanuel Dornier ◽  
Claude Boucheix ◽  
Stéphanie Charrin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cardiovascular Diseases ◽  
Cell Surface ◽  
Notch Signaling ◽  
Surface Proteins ◽  
Amyloid Peptide ◽  
Cell Surface Proteins ◽  
Β Amyloid ◽  
Β Amyloid Peptide ◽  
Partner Proteins

By interacting directly with partner proteins and with one another, tetraspanins organize a network of interactions referred to as the tetraspanin web. ADAM10 (A Disintegrin And Metalloprotease 10), an essential membrane-anchored metalloprotease that cleaves off the ectodomain of a large variety of cell surface proteins including cytokines, adhesion molecules, the precursor of the β-amyloid peptide APP or Notch, has emerged as a major component of the tetraspanin web. Recent studies have shown that ADAM10 associates directly with all members (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33) of a subgroup of tetraspanins having eight cysteines in the large extracellular domain and referred to as TspanC8. All TspanC8 regulate ADAM10 exit from the endoplasmic reticulum, but differentially regulate its subsequent trafficking and its function, and have notably a different impact on Notch signaling. TspanC8 orthologs in invertebrates also regulate ADAM10 trafficking and Notch signaling. It may be possible to target TspanC8 tetraspanins to modulate in a tissue- or substrate-restricted manner ADAM10 function in pathologies such as cardiovascular diseases, cancer or Alzheimer's disease.

scholarly journals Saccharomyces cerevisiae Rab-GDI Displacement Factor Ortholog Yip3p Forms Distinct Complexes with the Ypt1 Rab GTPase and the Reticulon Rtn1p

2005 ◽  
Vol 4 (7) ◽  
pp. 1166-1174 ◽  
Author(s):  
Jinming Geng ◽  
Marcus E. Shin ◽  
Penney M. Gilbert ◽  
Ruth N. Collins ◽  
Christopher G. Burd
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Complexes ◽  
Intracellular Localization ◽  
Integral Membrane Protein ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Organelle Biogenesis ◽  
Cell Extracts

ABSTRACT Rab GTPases are crucial regulators of organelle biogenesis, maintenance, and transport. Multiple Rabs are expressed in all cells, and each is localized to a distinct set of organelles, but little is known regarding the mechanisms by which Rabs are targeted to their resident organelles. Integral membrane proteins have been postulated to serve as receptors that recruit Rabs from the cytosol in a complex with the Rab chaperone, GDI, to facilitate the dissociation of Rab and GDI, hence facilitating loading of Rabs on membranes. We show here that the yeast (Saccharomyces cerevisiae) Golgi Rab GTPase Ypt1p can be copurified with the integral membrane protein Yip3p from detergent cell extracts. In addition, a member of the highly conserved reticulon protein family, Rtn1p, is also associated with Yip3p in vivo. However, Ypt1p did not copurify with Rtn1p, indicating that Yip3p is a component of at least two different protein complexes. Yip3p and Rtn1p are only partially colocalized in cells, with Yip3p localized predominantly to the Golgi and secondarily to the endoplasmic reticulum, whereas Rtn1p is localized predominantly to the endoplasmic reticulum and secondarily to the Golgi. Surprisingly, the intracellular localization of Rabs was not perturbed in yip3Δ or rtn1Δ mutants, suggesting that these proteins do not play a role in targeting Rabs to intracellular membranes. These data indicate that Yip3p may have multiple functions and that its interaction with Rabs is not critical for their recruitment to organelle membranes.

scholarly journals Actin-Dependent Nonlytic Rotavirus Exit and Infectious Virus Morphogenetic Pathway in Nonpolarized Cells

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Óscar Trejo-Cerro ◽  
Catherine Eichwald ◽  
Elisabeth M. Schraner ◽  
Daniela Silva-Ayala ◽  
Susana López ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Infectious Virus ◽  
Cell Attachment ◽  
Current Model ◽  
Intracellular Localization ◽  
Cell Lysis ◽  
Surface Proteins ◽  
Spike Protein

ABSTRACTDuring the late stages of rotavirus morphogenesis, the surface proteins VP4 and VP7 are assembled onto the previously structured double-layered virus particles to yield a triple-layered, mature infectious virus. The current model for the assembly of the outer capsid is that it occurs within the lumen of the endoplasmic reticulum. However, it has been shown that VP4 and infectious virus associate with lipid rafts, suggesting that the final assembly of the rotavirus spike protein VP4 involves a post-endoplasmic reticulum event. In this work, we found that the actin inhibitor jasplakinolide blocks the cell egress of rotavirus from nonpolarized MA104 cells at early times of infection, when there is still no evidence of cell lysis. These findings contrast with the traditional assumption that rotavirus is released from nonpolarized cells by a nonspecific mechanism when the cell integrity is lost. Inspection of the virus present in the extracellular medium by use of density flotation gradients revealed that a fraction of the released virus is associated with low-density membranous structures. Furthermore, the intracellular localization of VP4, its interaction with lipid rafts, and its targeting to the cell surface were shown to be prevented by jasplakinolide, implying a role for actin in these processes. Finally, the VP4 present at the plasma membrane was shown to be incorporated into the extracellular infectious virus, suggesting the existence of a novel pathway for the assembly of the rotavirus spike protein.IMPORTANCERotavirus is a major etiological agent of infantile acute severe diarrhea. It is a nonenveloped virus formed by three concentric layers of protein. The early stages of rotavirus replication, including cell attachment and entry, synthesis and translation of viral mRNAs, replication of the genomic double-stranded RNA (dsRNA), and the assembly of double-layered viral particles, have been studied widely. However, the mechanisms involved in the later stages of infection, i.e., viral particle maturation and cell exit, are less well characterized. It has been assumed historically that rotavirus exits nonpolarized cells following cell lysis. In this work, we show that the virus exits cells by a nonlytic, actin-dependent mechanism, and most importantly, we describe that VP4, the spike protein of the virus, is present on the cell surface and is incorporated into mature, infectious virus, indicating a novel pathway for the assembly of this protein.

scholarly journals Entamoeba histolytica Cell Surface Calreticulin Binds Human C1q and Functions in Amebic Phagocytosis of Host Cells

2012 ◽  
Vol 80 (6) ◽  
pp. 2008-2018 ◽  
Author(s):  
Archana Vaithilingam ◽  
Jose E. Teixeira ◽  
Peter J. Miller ◽  
Bradley T. Heron ◽  
Christopher D. Huston
Keyword(s):  
Cell Surface ◽  
Entamoeba Histolytica ◽  
Calcium Ionophore ◽  
Surface Expression ◽  
Surface Proteins ◽  
Host Cells ◽  
Content Type ◽  
Amebic Dysentery ◽  
Host Cell Apoptosis ◽  
Phagocytic Cups

ABSTRACTPhagocytosis of host cells is characteristic of tissue invasion by the intestinal amebaEntamoeba histolytica, which causes amebic dysentery and liver abscesses.Entamoeba histolyticainduces host cell apoptosis and uses ligands, including C1q, on apoptotic cells to engulf them. Two mass spectrometry analyses identified calreticulin in amebic phagosome preparations, and, in addition to its function as an endoplasmic reticulum chaperone, calreticulin is believed to be the macrophage receptor for C1q. The purpose of this study was to determine if calreticulin functions as anE. histolyticaC1q receptor during phagocytosis of host cells. Calreticulin was localized to the surface ofE. histolyticaduring interaction with both Jurkat lymphocytes and erythrocytes and was present in over 75% of phagocytic cups during amebic erythrophagocytosis. Presence of calreticulin on the cell surface was further demonstrated using a method that selectively biotinylated cell surface proteins and by flow cytometry using trophozoites overexpressing epitope-tagged calreticulin. Regulated overexpression of calreticulin increasedE. histolytica's ability to phagocytose apoptotic lymphocytes and calcium ionophore-treated erythrocytes but had no effect on amebic adherence to or destruction of cell monolayers or surface expression of the GalNAc lectin and serine-richE. histolyticaprotein (SREHP) receptors. Finally,E. histolyticacalreticulin bound specifically to apoptotic lymphocytes and to human C1q. Collectively, these data implicate cell surface calreticulin as a receptor for C1q duringE. histolyticaphagocytosis of host cells.

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