Evidence for a Vacuolar-Type Proton ATPase in Entamoeba histolytica

Abstract Entamoeba histolytica Entamoeba histolytica is a primitive eukaryote that lacks mitochondria, Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes. Similar to the latter, the amebal vesicles are acidified. We report here that bafilomycin AI, a specific inhibitor of vacuolar-type (V-) ATPases, suppressed this acidification at submicromolar concentrations; concom itantly, it inhibited pinocytosis. These results strongly suggest the presence of a V-ATPase in pinocytic vesicles of E. histolytica, and thereby support the notion that the V-ATPases in the organelles of higher eukaryotes are derived from an archaic plasma membrane-bound form.

Download Full-text

Specific Heterodimer Formation Is a Prerequisite for Uroplakins to Exit from the Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e02-04-0211 ◽

2002 ◽

Vol 13 (12) ◽

pp. 4221-4230 ◽

Cited By ~ 85

Author(s):

Liyu Tu ◽

Tung-Tien Sun ◽

Gert Kreibich

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Posttranslational Modifications ◽

Cell Layer ◽

Building Blocks ◽

Apical Plasma Membrane ◽

Membrane Bound ◽

Umbrella Cells ◽

Vesicular Compartment ◽

Lower Urinary

Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER.

Download Full-text

Studies of the endoplasmic reticulum and plasma membrane-bound ribosomes in erythropoietic cells

Journal of Cell Science ◽

10.1242/jcs.31.1.165 ◽

1978 ◽

Vol 31 (1) ◽

pp. 165-178

Author(s):

J.A. Grasso ◽

A.L. Sullivan ◽

S.C. Chan

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Exact Role ◽

Whole Cells ◽

Cytoplasmic Face ◽

Close Proximity ◽

Haem Biosynthesis ◽

Hypotonic Buffer ◽

Membrane Bound ◽

Hypotonic Lysis

Erythropoietic cells of 5 species, including man, contain endoplasmic reticulum present as individual cisternae or tubules scattered throughout the cytoplasm of all stages except mature RBCs. The endoplasmic reticulum is mainly agranular but occurs frequently as a variant of granular ER which is characterized by an asymmetrical and irregular distribution of ribosomes along one cytoplasmic face. In most cells, the endoplasmic reticulum occurs in close proximity to mitochondria or the plasma membrane, suggesting that the organelle may be involved in functions related to these structures, e.g. haem biosynthesis. Endoplasmic reticulum is more abundant in early than in late erythroid cells. Its exact role in RBC development is unclear. Since endoplasmic reticulum could account for ‘plasma membrane-bound ribosomes’ reported in lysed reticulocytes, studies were performed which ruled out this possibility and which suggested that such ribosomes were an artifact of the lysing conditions. Hypotonic lysis in less than 20 vol. of magnesium-containing buffers yielded ghosts variably contaminated by ribosomes and other structures. Lysis of reticulocytes in 20–30 vol. of magnesium-free buffer or homogenization of whole cells or crude membrane fractions in hypotonic buffer removed virtually all contaminating ribosomes from the purified membrane fraction.

Download Full-text

Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

Biochemical Journal ◽

10.1042/bj1520291 ◽

1975 ◽

Vol 152 (2) ◽

pp. 291-302 ◽

Cited By ~ 35

Author(s):

Richard Harwood ◽

Michael E. Grant ◽

David S. Jackson

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Specific Activity ◽

Smooth Endoplasmic Reticulum ◽

Gradient Centrifugation ◽

Subcellular Fractions ◽

Anterior Lens Capsule ◽

Cartilage Cells ◽

Membrane Bound ◽

And Cartilage

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [14C]lysine were assayed for hydroxy[14C]lysine and hydroxy[14C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[14C]lysine and glucosylgalactosylhydroxy[14C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [14C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. 14C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5′-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

Download Full-text

The Arabidopsis AtGCD3 protein is a glucosylceramidase that preferentially hydrolyzes long-acyl-chain glucosylceramides

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011274 ◽

2019 ◽

Vol 295 (3) ◽

pp. 717-728 ◽

Cited By ~ 2

Author(s):

Guang-Yi Dai ◽

Jian Yin ◽

Kai-En Li ◽

Ding-Kang Chen ◽

Zhe Liu ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Fusion Proteins ◽

Biosynthetic Pathway ◽

Acyl Chain ◽

Specific Inhibitor ◽

Endoplasmic Reticulum Membrane ◽

Growth Defects ◽

The Common

Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

Download Full-text

Trafficking Unconventionally via UPS

Cells ◽

10.3390/cells9092009 ◽

2020 ◽

Vol 9 (9) ◽

pp. 2009

Author(s):

Bor Luen Tang

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Proteins ◽

Golgi Apparatus ◽

Protein Secretion ◽

Vesicular Trafficking ◽

Eukaryotic Cells

Conventional protein secretion in eukaryotic cells occurs via vesicular trafficking of proteins that are first targeted to the endoplasmic reticulum (ER), through the Golgi apparatus, and subsequently routed to the plasma membrane (PM), where membrane proteins take up residence while luminal proteins are released extracellularly [...]

Download Full-text

Tropomodulins and tropomyosins – organizers of cellular microcompartments

BioMolecular Concepts ◽

10.1515/bmc-2012-0037 ◽

2013 ◽

Vol 4 (1) ◽

pp. 89-101 ◽

Cited By ~ 3

Author(s):

Thomas Fath

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Critical Role ◽

Eukaryotic Cells ◽

Multigene Families ◽

Cellular Functions ◽

Cellular Morphogenesis ◽

Wide Range ◽

Associated Proteins

AbstractEukaryotic cells show a remarkable compartmentalization into compartments such as the cell nucleus, the Golgi apparatus, the endoplasmic reticulum, and endosomes. However, organelle structures are not the only means by which specialized compartments are formed. Recent research shows a critical role for diverse actin filament populations in defining functional compartments, here referred to as microcompartments, in a wide range of cells. These microcompartments are involved in regulating fundamental cellular functions including cell motility, plasma membrane organization, and cellular morphogenesis. In this overview, the importance of two multigene families of actin-associated proteins, tropomodulins and tropomyosins, their interactions with each other, and a large number of other proteins will be discussed in the context of generating specialized actin-based microcompartments.

Download Full-text

A NET4-RabG3 couple mediate the link between actin and the tonoplast and is essential for normal actin cytoskeletal remodelling in stomatal closure to flg22

10.1101/2021.09.29.461190 ◽

2021 ◽

Author(s):

Timothy J HAWKINS ◽

Michaela Kopischke ◽

David Mentlak ◽

Patrick Duckney ◽

Johan Kroon ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Guard Cell ◽

Actin Filaments ◽

Plant Immunity ◽

Stomatal Closure ◽

Actin Network ◽

Actin Reorganization ◽

Downstream Effectors ◽

Membrane Bound

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. They tether the cell's plasma membrane (PM) to the actin network. Moreover, in a similar manner, they are also involved in the tethering of membrane bound organelles to the actin cytoskeleton; the endoplasmic reticulum (ER) and the ER to the PM. This raises the question as to whether NET proteins are involved in actin cytoskeletal remodelling. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function as downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure depends on a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune signalling events and proper actin cytoskeletal remodelling.

Download Full-text

Ultrastructure of dormant basidiospores of Agaricus campestris

Canadian Journal of Botany ◽

10.1139/b88-084 ◽

1988 ◽

Vol 66 (3) ◽

pp. 583-587 ◽

Cited By ~ 5

Author(s):

Donald G. Ruch ◽

Mary C. North

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Short Distance ◽

Mitochondrial Membrane ◽

Lipid Droplets ◽

Wall Material ◽

Outer Mitochondrial Membrane ◽

The Third ◽

Membrane Bound ◽

Agaricus Campestris

The basidiospore wall of Agaricus campestris Fr. consists of three distinct layers. The outer two layers are continuous around the spore, while the third layer originates only a short distance from the hilar appendage and quickly thickens to form the bulk of the wall material of the hilar appendage. The protoplast is surrounded by a typical plasma membrane which lacks distinct invaginations. Centrally located nonmembrane-bound lipid droplets comprise the bulk of the protoplasm. Spores are binucleate, but the two nuclei do not exhibit any distinct relationship to each other. Sausage-shaped mitochondria with only a few but well-delineated plate-like cristae are present. Scant endoplasmic reticulum occurs just beneath the plasma membrane. Ribosomes occur regularly attached to the endoplasmic reticulum and outer mitochondrial membrane, as well as being densely packed throughout the cytoplasm. The structure and possible functions of single membrane bound vacuoles and microbody-like organelles are discussed in relation to other basidiospores.

Download Full-text

How Viruses Hijack and Modify the Secretory Transport Pathway

Cells ◽

10.3390/cells10102535 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2535

Author(s):

Zubaida Hassan ◽

Nilima Dinesh Kumar ◽

Fulvio Reggiori ◽

Gulfaraz Khan

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Eukaryotic Cells ◽

Environmental Cues ◽

Transport Pathway ◽

Dynamic Membrane ◽

Enveloped Viruses ◽

Vesicular Traffic ◽

Secretory Transport ◽

Membrane Bound

Eukaryotic cells contain dynamic membrane-bound organelles that are constantly remodeled in response to physiological and environmental cues. Key organelles are the endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which are interconnected by vesicular traffic through the secretory transport route. Numerous viruses, especially enveloped viruses, use and modify compartments of the secretory pathway to promote their replication, assembly and cell egression by hijacking the host cell machinery. In some cases, the subversion mechanism has been uncovered. In this review, we summarize our current understanding of how the secretory pathway is subverted and exploited by viruses belonging to Picornaviridae, Coronaviridae, Flaviviridae, Poxviridae, Parvoviridae and Herpesviridae families.

Download Full-text

Differential permeabilization of membranes by saponin treatment of isolated rat hepatocytes. Release of secretory proteins

Biochemical Journal ◽

10.1042/bj2470407 ◽

1987 ◽

Vol 247 (2) ◽

pp. 407-415 ◽

Cited By ~ 69

Author(s):

M Wassler ◽

I Jonasson ◽

R Persson ◽

E Fries

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Lactate Dehydrogenase ◽

Rat Hepatocytes ◽

Secretory Proteins ◽

Isolated Rat Hepatocytes ◽

Monolayer Cultures ◽

Intracellular Compartments ◽

Membrane Bound ◽

Isolated Rat

Monolayer cultures of rat hepatocytes were treated with increasing concentrations of saponin (prepared from Gypsophila plants) for 30 min at 6 degrees C. Differential permeabilization of the intracellular membranes could be demonstrated: at 0.040 mg of saponin/ml the plasma membrane was permeabilized, as assessed by the release of 50% of the total cellular amount of lactate dehydrogenase, and at 0.20 mg/ml the endoplasmic reticulum was permeabilized, as measured by the release of 50% of pulse-35S-labelled albumin. The Golgi complex was permeabilized at an intermediate saponin concentration, as indicated by the release of homogeneously 35S-labelled albumin; about half the intracellular albumin is located in this organelle. At 1.0 up to 5.0 mg of saponin/ml 90-95% of the radioactively labelled albumin was released. Even at 5.0 mg/ml less than 10% of the membrane of the endoplasmic reticulum was solubilized, as judged by the degree of release of a membrane-bound enzyme specific for this organelle. These results demonstrate the usefulness of saponin as a tool for investigating the interior of different intracellular compartments.

Download Full-text