scholarly journals Endocytosis of GPI-linked membrane folate receptor-alpha.

1996 ◽  
Vol 132 (1) ◽  
pp. 35-47 ◽  
Author(s):  
S Rijnboutt ◽  
G Jansen ◽  
G Posthuma ◽  
J B Hynes ◽  
J H Schornagel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Folate Receptor ◽  
Large Fraction ◽  
Integral Membrane Protein ◽  
Endocytic Pathway ◽  
Reduced Folate Carrier ◽  
Membrane Domains ◽  
Chemotherapeutic Drugs ◽  
Folate Receptors ◽  
Triton X

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

scholarly journals An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

1998 ◽  
Vol 9 (3) ◽  
pp. 599-609 ◽  
Author(s):  
Hans de Vries ◽  
Cobi Schrage ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Insoluble Fraction ◽  
Membrane Domains ◽  
Myelin Membrane ◽  
Influenza Virus Hemagglutinin ◽  
Polarized Cells ◽  
Vesicular Stomatitis ◽  
Triton X

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus–hemagglutinin (HA) and vesicular stomatitis virus–G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.

scholarly journals Cytoskeleton-dependent Membrane Domain Segregation during Neutrophil Polarization

2001 ◽  
Vol 12 (11) ◽  
pp. 3550-3562 ◽  
Author(s):  
Stéphanie Seveau ◽  
Robert J. Eddy ◽  
Frederick R. Maxfield ◽  
Lynda M. Pierini
Keyword(s):  
Plasma Membrane ◽  
Myosin Light Chain ◽  
Transmembrane Proteins ◽  
Cell Polarization ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Actin Remodeling ◽  
Cytoskeletal Rearrangements ◽  
Triton X ◽  
Detergent Resistant Membrane

On treatment with chemoattractant, the neutrophil plasma membrane becomes organized into detergent-resistant membrane domains (DRMs), the distribution of which is intimately correlated with cell polarization. Plasma membrane at the front of polarized cells is susceptible to extraction by cold Triton X-100, whereas membrane at the rear is resistant to extraction. After cold Triton X-100 extraction, DRM components, including the transmembrane proteins CD44 and CD43, the GPI-linked CD16, and the lipid analog, DiIC16, are retained within uropods and cell bodies. Furthermore, CD44 and CD43 interact concomitantly with DRMs and with the F-actin cytoskeleton, suggesting a mechanism for the formation and stabilization of DRMs. By tracking the distribution of DRMs during polarization, we demonstrate that DRMs progress from a uniform distribution in unstimulated cells to small, discrete patches immediately after activation. Within 1 min, DRMs form a large cap comprising the cell body and uropod. This process is dependent on myosin in that an inhibitor of myosin light chain kinase can arrest DRM reorganization and cell polarization. Colabeling DRMs and F-actin revealed a correlation between DRM distribution and F-actin remodeling, suggesting that plasma membrane organization may orient signaling events that control cytoskeletal rearrangements and, consequently, cell polarity.

scholarly journals The Major Myelin-Resident Protein PLP Is Transported to Myelin Membranes via a Transcytotic Mechanism: Involvement of Sulfatide

2014 ◽  
Vol 35 (1) ◽  
pp. 288-302 ◽  
Author(s):  
Wia Baron ◽  
Hande Ozgen ◽  
Bert Klunder ◽  
Jenny C. de Jonge ◽  
Anita Nomden ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Body ◽  
Proteolipid Protein ◽  
Membrane Microdomains ◽  
Apical Surface ◽  
Membrane Domains ◽  
Myelin Membrane ◽  
Conformational Alteration ◽  
Syntaxin 3 ◽  
Triton X

Myelin membranes are sheet-like extensions of oligodendrocytes that can be considered membrane domains distinct from the cell's plasma membrane. Consistent with the polarized nature of oligodendrocytes, we demonstrate that transcytotic transport of the major myelin-resident protein proteolipid protein (PLP) is a key element in the mechanism of myelin assembly. Upon biosynthesis, PLP traffics to myelin membranes via syntaxin 3-mediated docking at the apical-surface-like cell body plasma membrane, which is followed by subsequent internalization and transport to the basolateral-surface-like myelin sheet. Pulse-chase experiments, in conjunction with surface biotinylation and organelle fractionation, reveal that following biosynthesis, PLP is transported to the cell body surface in Triton X-100 (TX-100)-resistant microdomains. At the plasma membrane, PLP transiently resides within these microdomains and its lateral dissipation is followed by segregation into 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)-resistant domains, internalization, and subsequent transport toward the myelin membrane. Sulfatide triggers PLP's reallocation from TX-100- into CHAPS-resistant membrane domains, while inhibition of sulfatide biosynthesis inhibits transcytotic PLP transport. Taking these findings together, we propose a model in which PLP transport to the myelin membrane proceeds via a transcytotic mechanism mediated by sulfatide and characterized by a conformational alteration and dynamic, i.e., transient, partitioning of PLP into distinct membrane microdomains involved in biosynthetic and transcytotic transport.

scholarly journals VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles.

1992 ◽  
Vol 118 (5) ◽  
pp. 1003-1014 ◽  
Author(s):  
T V Kurzchalia ◽  
P Dupree ◽  
R G Parton ◽  
R Kellner ◽  
H Virta ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Cellular Membrane ◽  
Integral Membrane Proteins ◽  
Membrane Domains ◽  
Transport Vesicles ◽  
Vesicular Carriers ◽  
Molecular Machinery ◽  
Golgi Network

In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.

Folate Receptors in Malignant and Benign Tissues of Human Female Genital Tract

1997 ◽  
Vol 17 (4) ◽  
pp. 415-427 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen ◽  
Poul Erik Helkjær ◽  
Carl W. Nichols
Keyword(s):  
Genital Tract ◽  
Folate Receptor ◽  
Female Genital Tract ◽  
Gel Filtration ◽  
Human Female ◽  
Hydrophobic Membrane ◽  
Folate Receptors ◽  
High Affinity ◽  
Triton X ◽  
Female Genital

We have characterized the folate receptor in malignant and benign tissues of human female genital tract (Fallopian tube and benign and malignant tissues of uterus). Radioligand binding displayed characteristics similar to those of other folate binding proteins. Those include a high-affinity type of binding (K=1010M−1), apparent positive cooperativity, a slow dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition of binding by folate analogues. The gel filtration profile of Triton X-100 solubilized tissue contained two large peaks of 3H-folate labelled protein (>=130 and 100kDa) as well as a 25 kDa peak. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The large molecular size forms on gel filtration appear to represent folate receptors having a hydrophobic membrane anchor inserted into Triton X-100 micelles. The folate receptor of female genital tract showed cross-reactivity in ELISA and positive immunostaining with rabbit antibodies against human milk folate binding protein. Variations in the ratio of immunoresponse to total high affinity folic acid binding suggests the presence of multiple isoforms of the receptor in different types of malignant and benign tissues.

scholarly journals p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

2009 ◽  
Vol 20 (7) ◽  
pp. 1970-1980 ◽  
Author(s):  
Christine M. Chiasson ◽  
Kristin B. Wittich ◽  
Peter A. Vincent ◽  
Victor Faundez ◽  
Andrew P. Kowalczyk
Keyword(s):  
Plasma Membrane ◽  
Rho Gtpase ◽  
Retention Mechanism ◽  
Endocytic Pathway ◽  
Membrane Domains ◽  
P120 Catenin ◽  
Rhoa Activity ◽  
Endocytic Machinery ◽  
Endocytic Compartments ◽  
The Relationship

p120-catenin is a cytoplasmic binding partner of cadherins and functions as a set point for cadherin expression by preventing cadherin endocytosis, and degradation. p120 is known to regulate cell motility and invasiveness by inhibiting RhoA activity. However, the relationship between these functions of p120 is not understood. Here, we provide evidence that p120 functions as part of a plasma membrane retention mechanism for VE-cadherin by preventing the recruitment of VE-cadherin into membrane domains enriched in components of the endocytic machinery, including clathrin and the adaptor complex AP-2. The mechanism by which p120 regulates VE-cadherin entry into endocytic compartments is dependent on p120's interaction with the cadherin juxtamembrane domain, but occurs independently of p120's prevention of Rho GTPase activity. These findings clarify the mechanism for p120's function in stabilizing VE-cadherin at the plasma membrane and demonstrate a novel role for p120 in modulating the availability of cadherins for entry into a clathrin-dependent endocytic pathway.

CD44 exhibits a cell type dependent interaction with triton X-100 insoluble, lipid rich, plasma membrane domains

1995 ◽  
Vol 108 (9) ◽  
pp. 3127-3135 ◽  
Author(s):  
S.J. Neame ◽  
C.R. Uff ◽  
H. Sheikh ◽  
S.C. Wheatley ◽  
C.M. Isacke
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Direct Interaction ◽  
Membrane Domains ◽  
Cell Type ◽  
Transmembrane Glycoprotein ◽  
Morphological Studies ◽  
Plasma Membrane Domains ◽  
Triton X

CD44 is an abundant, widely expressed transmembrane glycoprotein which can act as a receptor for the extracellular matrix glycosaminoglycan, hyaluronan. Biochemical and morphological studies have demonstrated that in fibroblasts a significant of the CD44 population is resistant to Triton X-100 extraction and that the detergent insoluble protein is co-localized with components of the cortical cytoskeleton. Surprisingly, this distribution is not abrogated upon deletion of the CD44 cytoplasmic tail indicating that mechanisms other than a direct interaction with the cytoskeleton can regulate CD44. In this manuscript, the mechanisms underlying this detergent-insoluble association are further investigated. There was no evidence that the Triton X-100 insolubility of CD44 resulted from homotypic aggregation, an association with hyaluronan or from a direct, or indirect, association with the cytoskeleton. Instead, evidence is presented that the detergent insolubility of fibroblast CD44 at 4 degrees C results from an association of the CD44 transmembrane domain with Triton X-100 resistant, lipid rich, plasma membrane domains. The proportion of the CD44 found in these Triton X-100 insoluble structures is dependent upon cell type and cannot be altered by changing cell motility or extracellular matrix associations. These studies provide evidence for a novel mechanism regulating this adhesion protein in the plasma membrane.

scholarly journals Human Immunodeficiency Virus Type 1 Assembly and Lipid Rafts: Pr55gag Associates with Membrane Domains That Are Largely Resistant to Brij98 but Sensitive to Triton X-100

2003 ◽  
Vol 77 (8) ◽  
pp. 4805-4817 ◽  
Author(s):  
Kirsi Holm ◽  
Katarzyna Weclewicz ◽  
Roger Hewson ◽  
Maarit Suomalainen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Intracellular Membranes ◽  
Immunodeficiency Virus ◽  
Triton X

ABSTRACT The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55 gag . We have analyzed whether Pr55 gag has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55 gag has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55 gag particles (VLPs) yield buoyant Pr55 gag complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55gag complexes cannot be taken as a proof for raft association of Pr55 gag , since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55 gag . However, Pr55 gag might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55 gag . Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55 gag complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55 gag localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55 gag VLPs.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

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