Chicken Erythroid AE1 Anion Exchangers Associate with the Cytoskeleton During Recycling to the Golgi

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

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Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

Journal of Cell Science ◽

10.1242/jcs.103.4.1139 ◽

1992 ◽

Vol 103 (4) ◽

pp. 1139-1152

Author(s):

J.W. Kok ◽

K. Hoekstra ◽

S. Eskelinen ◽

D. Hoekstra

Keyword(s):

Plasma Membrane ◽

Intracellular Ph ◽

Brefeldin A ◽

Fluid Phase ◽

Endocytic Pathway ◽

Late Endosomes ◽

Early Endosomes ◽

Recycling Pathway ◽

Golgi Network ◽

Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

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Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

Molecular Biology of the Cell ◽

10.1091/mbc.e11-11-0944 ◽

2012 ◽

Vol 23 (12) ◽

pp. 2339-2351 ◽

Cited By ~ 46

Author(s):

Yogikala Prabhu ◽

Patricia V. Burgos ◽

Christina Schindler ◽

Ginny G. Farías ◽

Javier G. Magadán ◽

...

Keyword(s):

Plasma Membrane ◽

Amyloid Precursor Protein ◽

Secretory Pathway ◽

Brefeldin A ◽

Adaptor Protein ◽

Proteolytic Processing ◽

Precursor Protein ◽

Amyloid Precursor Protein Processing ◽

Intermediate Compartment ◽

Golgi Network

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.

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Palmitoylation and Plasma Membrane Localization of Ras2p by a Nonclassical Trafficking Pathway in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.23.18.6574-6584.2003 ◽

2003 ◽

Vol 23 (18) ◽

pp. 6574-6584 ◽

Cited By ~ 55

Author(s):

Xiangwen Dong ◽

David A. Mitchell ◽

Sandra Lobo ◽

Lihong Zhao ◽

Douglas J. Bartels ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Secretory Pathway ◽

Brefeldin A ◽

Covalent Attachment ◽

Membrane Localization ◽

Ras Proteins ◽

Plasma Membrane Localization ◽

Additional Processing ◽

Endomembrane Trafficking

ABSTRACT Subcellular localization of Ras proteins to the plasma membrane is accomplished in part by covalent attachment of a farnesyl moiety to the conserved CaaX box cysteine. Farnesylation targets Ras to the endoplasmic reticulum (ER), where additional processing steps occur, resulting in translocation of Ras to the plasma membrane. The mechanism(s) by which this occurs is not well understood. In this report, we show that plasma membrane localization of Ras2p in Saccharomyces cerevisiae does not require the classical secretory pathway or a functional Golgi apparatus. However, when the classical secretory pathway is disrupted, plasma membrane localization requires Erf2p, a protein that resides in the ER membrane and is required for efficient palmitoylation of Ras2p. Deletion of ERF2 results in a Ras2p steady-state localization defect that is more severe when combined with sec-ts mutants or brefeldin A treatment. The Erf2p-dependent localization of Ras2p correlates with the palmitoylation of Cys-318. An Erf2p-Erf4p complex has recently been shown to be an ER-associated palmitoyltransferase that can palmitoylate Cys-318 of Ras2p (S. Lobo, W. K. Greentree, M. E. Linder, and R. J. Deschenes, J. Biol. Chem. 277:41268-41273, 2002). Erf2-dependent palmitoylation as well as localization of Ras2p requires a region of the hypervariable domain adjacent to the CaaX box. These results provide evidence for the existence of a palmitoylation-dependent, nonclassical endomembrane trafficking system for the plasma membrane localization of Ras proteins.

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Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

Molecular Biology of the Cell ◽

10.1091/mbc.8.1.13 ◽

1997 ◽

Vol 8 (1) ◽

pp. 13-31 ◽

Cited By ~ 76

Author(s):

L Hicke ◽

B Zanolari ◽

M Pypaert ◽

J Rohrer ◽

H Riezman

Keyword(s):

Fusion Protein ◽

Secretory Pathway ◽

Protein A ◽

Brefeldin A ◽

Early Endosome ◽

Late Endosome ◽

Endocytic Pathway ◽

Early Secretory Pathway ◽

Late Endosomes

Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.

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Stimulation of an N-ethylmaleimide-sensitive ATPase in the collecting duct segments of the rat nephron by metabolic acidosis

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y85-213 ◽

1985 ◽

Vol 63 (10) ◽

pp. 1291-1296 ◽

Cited By ~ 16

Author(s):

Lal C. Garg ◽

Neelam Narang

Keyword(s):

Enzyme Activity ◽

Plasma Membrane ◽

Metabolic Acidosis ◽

Ammonium Chloride ◽

Atp Hydrolysis ◽

Collecting Duct ◽

Plasma Membrane Atpase ◽

Membrane Atpase ◽

Chloride Treatment ◽

Stimulation Of

A plasma membrane ATPase sensitive to inhibition by N-ethylmaleimide (NEM) and insensitive to inhibition by oligomycin and ouabain has been shown to be involved in acidification of urine in the turtle bladder. The activity of this NEM-sensitive ATPase was determined in four types of distal nephron segments of normal rats and in rats treated with ammonium chloride. The enzyme activity was determined by a fluorometric micromethod in which ATP hydrolysis was coupled to NADH oxidation. Significant activities (10–35 pmol ADP∙min−1∙mm−1) of NEM-sensitive ATPase were present in the distal convoluted tubule (DCT) and in the cortical and outer and inner medullary collecting duet segments of normal rats. In metabolic acidosis produced by ammonium chloride treatment (plasma CO2 content = 15.3 ± 0.8 mequiv./L), the NEM-sensitive ATPase activity was increased significantly (60–100%) in the collecting duct segments without showing a significant change in the enzyme activity in the DCT. Our data are consistent with the hypothesis that a plasma membrane H+-ATPase (inhibited by NEM but not by oligomycin or ouabain) is involved in H+ secretion in the mammalian collecting duct.

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The Function of the Intermediate Compartment in Pre-Golgi Trafficking Involves its Stable Connection with the Centrosome

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1229 ◽

2009 ◽

Vol 20 (20) ◽

pp. 4458-4470 ◽

Cited By ~ 35

Author(s):

Michaël Marie ◽

Hege A. Dale ◽

Ragna Sannerud ◽

Jaakko Saraste

Keyword(s):

Secretory Pathway ◽

Focal Point ◽

Brefeldin A ◽

Temperature Sensitive ◽

Cell Periphery ◽

Functional Connection ◽

Centrosome Separation ◽

Membrane Compartment ◽

Intermediate Compartment ◽

Endosomal System

Because the functional borders of the intermediate compartment (IC) are not well defined, the spatial map of the transport machineries operating between the endoplasmic reticulum (ER) and the Golgi apparatus remains incomplete. Our previous studies showed that the IC consists of interconnected vacuolar and tubular parts with specific roles in pre-Golgi trafficking. Here, using live cell imaging, we demonstrate that the tubules containing the GTPase Rab1A create a long-lived membrane compartment around the centrosome. Separation of this pericentrosomal domain of the IC from the Golgi ribbon, due to centrosome motility, revealed that it contains a distinct pool of COPI coats and acts as a temperature-sensitive way station in post-ER trafficking. However, unlike the Golgi, the pericentrosomal IC resists the disassembly of COPI coats by brefeldin A, maintaining its juxtaposition with the endocytic recycling compartment, and operation as the focal point of a dynamic tubular network that extends to the cell periphery. These results provide novel insight into the compartmental organization of the secretory pathway and Golgi biogenesis. Moreover, they reveal a direct functional connection between the IC and the endosomal system, which evidently contributes to unconventional transport of the cystic fibrosis transmembrane conductance regulator to the cell surface.

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Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane.

The Journal of Cell Biology ◽

10.1083/jcb.123.6.1403 ◽

1993 ◽

Vol 123 (6) ◽

pp. 1403-1419 ◽

Cited By ~ 48

Author(s):

L S Kean ◽

R S Fuller ◽

J W Nichols

Keyword(s):

Plasma Membrane ◽

Fluorescence Microscopy ◽

Nuclear Envelope ◽

Secretory Pathway ◽

Acyl Chain ◽

Energy Charge ◽

Endocytic Pathway ◽

Atp Depletion ◽

Yeast Saccharomyces Cerevisiae ◽

Flip Flop

Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Two pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature (< 2 degrees C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mutations in the SEC63, SEC65, or SEC11 genes, required for translocation of nascent secretory polypeptides into the ER or signal peptide processing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin heavy chain, CHC1. The correlation of biochemical analysis with fluorescence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, dependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced) and was largely independent of the SEC genes. Non-endocytic transport of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzenesulfonic acid, suggesting the existence of protein-mediated transmembrane transfer (flip-flop) of phosphatidylcholine across the yeast plasma membrane. These data establish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracellular organelles through non-endocytic, protein-mediated events.

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Dilatation of Golgi vesicles by monensin leads to enhanced accumulation of sugar nucleotides

Bioscience Reports ◽

10.1007/bf01115011 ◽

1986 ◽

Vol 6 (2) ◽

pp. 227-234 ◽

Cited By ~ 8

Author(s):

Roméo Cecchelli ◽

René Cacan ◽

Eliane Porchet-Hennere ◽

André Verbert

Keyword(s):

Plasma Membrane ◽

Ammonium Chloride ◽

Direct Effect ◽

Golgi Complex ◽

Sugar Nucleotides ◽

Morphological Alterations ◽

Golgi Vesicles ◽

Intracellular Vesicles ◽

Chloride Treatment

Incubation of mouse thymocytes with 10μM monensin for 1 hour induces morphological alterations characterized by the extensive dilatation and vacuolization of the Golgi complex. This effect is used to study the transport and utilization of labelled sugar nucleotides into intracellular vesicles by using thymocytes whose plasma membrane has been permeabilized by ammonium chloride treatment. It is demonstrated that monensin stimulates the incorporation of labelled sialyl, fucosyl, galactosyl, and N-acetylglucosaminyl residues. This enhanced incorporation is not due to a direct effect of monensin on glycosyltransferase activities themselves but is a consequence of a higher entry and accumulation of labelled sugar nucleotides in the dilated vesicles.

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Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.

The Journal of Cell Biology ◽

10.1083/jcb.128.6.1003 ◽

1995 ◽

Vol 128 (6) ◽

pp. 1003-1017 ◽

Cited By ~ 259

Author(s):

P J Peters ◽

V W Hsu ◽

C E Ooi ◽

D Finazzi ◽

S B Teal ◽

...

Keyword(s):

Plasma Membrane ◽

Golgi Complex ◽

Secretory Pathway ◽

Membrane System ◽

Endocytic Pathway ◽

Coat Proteins ◽

Wild Type ◽

Gtp Binding ◽

Defective Mutant

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

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Chs6p-dependent Anterograde Transport of Chs3p from the Chitosome to the Plasma Membrane inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.9.6.1565 ◽

1998 ◽

Vol 9 (6) ◽

pp. 1565-1576 ◽

Cited By ~ 107

Author(s):

Michael Ziman ◽

John S. Chuang ◽

Michael Tsung ◽

Susan Hamamoto ◽

Randy Schekman

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Cell Surface ◽

Cell Separation ◽

Secretory Pathway ◽

Protein Transport ◽

Subcellular Fractionation ◽

Chitin Synthase ◽

Anterograde Transport ◽

Membrane Compartment

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Δ strains have morphological abnormalities similar to those of chs3mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6mutant cells. Order-of-function experiments usingend4–1 (endocytosis-defective) and chs6mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.

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