Maturation of reticulocytes: formation of exosomes as a mechanism for shedding membrane proteins

1992 ◽  
Vol 70 (3-4) ◽  
pp. 179-190 ◽  
Author(s):  
R. M. Johnstone
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Structural Changes ◽  
Surface Proteins ◽  
Multivesicular Bodies ◽  
Red Cell ◽  
Nucleoside Transporter ◽  
Selective Loss ◽  
Anion Transporter

The transferrin receptor is a member of a group of reticulocyte surface proteins that disappear from the membranes of reticulocytes as the cells mature to the erythrocyte stage. The selective loss of membrane proteins appears to be preceded by the formation of multivesicular bodies (MVBs). At the reticulocyte stage, many species of mammalian red cells including man, and one nucleated avian species (chicken), contain these intracellular structures in both natural and induced anemias. Also characteristic of blood containing reticulocytes is the presence of circulating vesicles (exosomes), which contain proteins and lipids characteristic of the plasma membrane. These exosomes appear to arise from the contents of the MVBs, after the fusion of MVBs with the plasma membrane. The proteins in the exosomes are those frequently lost during red cell maturation (e.g., transferrin receptor). The major transmembrane proteins (such as the anion transporter) are fully retained into the mature red cell, indicating a highly selective mechanism of recognition of a specific group of proteins. The exosomes are largely devoid of soluble proteins and proteins associated with lysozomes or mitochondria. A speculative model is proposed which addresses the questions of the maturation-induced structural changes in a class of membrane proteins, their recognition and selective loss involving exosome formation, and the release of exosomes to the circulation.Key words: transferrin receptor, nucleoside transporter, reticulocyte maturation, multivesicular bodies, 70-kilodalton protein.

Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1844-1851 ◽  
Author(s):  
RM Johnstone ◽  
A Bianchini ◽  
K Teng
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Magnetic Core ◽  
Common Mechanism ◽  
Nucleoside Transporter ◽  
Plasma Membrane Proteins ◽  
Reticulocyte Maturation

Vesicles (exosomes) released during sheep reticulocyte maturation contain a number of plasma membrane functions. Using an antibody coated, magnetic core bead, it has been shown unequivocally that vesicles that contain the transferrin receptor also contain other plasma membrane activities, such as the nucleoside transporter and acetylcholinesterase. Lysosomal activities, normally found in the same pellet, are excluded from the transferrin receptor-containing exosomes, suggesting that there is a common mechanism to segregate and externalize specific plasma membrane proteins. In addition to the sheep, electron micrographic studies show that exosomes can be retrieved from the circulation of anemic pigs, rats, and rabbits.

scholarly journals Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1844-1851 ◽  
Author(s):  
RM Johnstone ◽  
A Bianchini ◽  
K Teng
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Magnetic Core ◽  
Common Mechanism ◽  
Nucleoside Transporter ◽  
Plasma Membrane Proteins ◽  
Reticulocyte Maturation

Abstract Vesicles (exosomes) released during sheep reticulocyte maturation contain a number of plasma membrane functions. Using an antibody coated, magnetic core bead, it has been shown unequivocally that vesicles that contain the transferrin receptor also contain other plasma membrane activities, such as the nucleoside transporter and acetylcholinesterase. Lysosomal activities, normally found in the same pellet, are excluded from the transferrin receptor-containing exosomes, suggesting that there is a common mechanism to segregate and externalize specific plasma membrane proteins. In addition to the sheep, electron micrographic studies show that exosomes can be retrieved from the circulation of anemic pigs, rats, and rabbits.

scholarly journals Salmonella typhimurium induces selective aggregation and internalization of host cell surface proteins during invasion of epithelial cells

1994 ◽  
Vol 107 (7) ◽  
pp. 2005-2020 ◽  
Author(s):  
F. Garcia-del Portillo ◽  
M.G. Pucciarelli ◽  
W.A. Jefferies ◽  
B.B. Finlay
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Heavy Chain ◽  
Surface Proteins ◽  
Class I ◽  
Plasma Membrane Proteins ◽  
Class I Mhc ◽  
Host Plasma Membrane

Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, beta 2-microglobulin, fibronectin-receptor (alpha 5 beta 1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to beta 1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia-internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.

The exosome pathway in K562 cells is regulated by Rab11

2002 ◽  
Vol 115 (12) ◽  
pp. 2505-2515 ◽  
Author(s):  
Ariel Savina ◽  
Michel Vidal ◽  
Maria I. Colombo
Keyword(s):  
Transferrin Receptor ◽  
K562 Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Rab Proteins ◽  
Multivesicular Bodies ◽  
Red Cell ◽  
Cell Type ◽  
Extracellular Medium ◽  
Endosomal Membrane

During maturation, reticulocytes lose some membrane proteins that are not required on the mature red cell surface. The proteins are released into the extracellular medium associated with vesicles that are formed by budding of the endosomal membrane into the lumen of the compartment; this process results in the formation of multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles, termed exosomes. K562 cells release exosomes with similar characteristics to reticulocyte exosomes, in particular the transferrin receptor (TfR) is found associated with the vesicles. Interestingly, this cell line has been shown to possess high amounts of Rab11 compared with other Rab proteins. To assess the regulation of transferrin receptor release via exosome secretion by Rab11 in this cell type, K562 cells were stably transfected with GFP-Rab11wt or the GTP- and GDP-locked mutants. The distribution of the proteins was assessed by fluorescence microscopy. Transferrin recycling and the number of TfRs present on the surface of the transfected cells were reduced by overexpression of either Rab11wt or the mutants. The amount of released exosomes was analyzed by measuring different molecular markers present on these vesicles either biochemically or by western blot. Overexpression of the dominant-negative mutant Rab11S25N inhibited exosome release, whereas the secretion of exosomes was slightly stimulated in cells transfected with Rab11wt. Taken together, the results demonstrate that in K562 cells Rab11 modulates the exosome pathway although the exact step involved is still not known.

scholarly journals Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins via Tubulovesicular Recycling Endosomes

1998 ◽  
Vol 143 (4) ◽  
pp. 957-971 ◽  
Author(s):  
Rytis Prekeris ◽  
Judith Klumperman ◽  
Yu A. Chen ◽  
Richard H. Scheller
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Trafficking ◽  
Transferrin Receptor ◽  
Endosomal Trafficking ◽  
Plasma Membrane Proteins ◽  
Recycling Endosomes ◽  
Confocal Immunofluorescence ◽  
Triton X ◽  
Coated Membrane

Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of βSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added αSNAP and NSF and dissociates in the presence of ATP, but not ATPγS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

Transferrin receptor 2 mediates a biphasic pattern of transferrin uptake associated with ligand delivery to multivesicular bodies

2004 ◽  
Vol 287 (6) ◽  
pp. C1769-C1775 ◽  
Author(s):  
Aeisha D. Robb ◽  
Maria Ericsson ◽  
Marianne Wessling-Resnick
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Physiological Role ◽  
Cell Types ◽  
Multivesicular Bodies ◽  
Unique Role ◽  
Biphasic Pattern ◽  
Exogenous Expression ◽  
Transferrin Uptake

The physiological role of transferrin (Tf) receptor 2 (TfR2), a homolog of the well-characterized TfR1, is unclear. Mutations in TfR2 result in hemochromatosis, indicating that this receptor has a unique role in iron metabolism. We report that HepG2 cells, which endogenously express TfR2, display a biphasic pattern of Tf uptake when presented with ligand concentrations up to 2 μM. The apparently nonsaturating pathway of Tf endocytosis resembles TfR1-independent Tf uptake, a process previously characterized in some liver cell types. Exogenous expression of TfR2 but not TfR1 induces a similar biphasic pattern of Tf uptake in HeLa cells, supporting a role for TfR2 in this process. Immunoelectron microscopy reveals that while Tf, TfR1, and TfR2 are localized in the plasma membrane and tubulovesicular endosomes, TfR2 expression is associated with the additional appearance of Tf in multivesicular bodies. These combined results imply that unlike TfR1, which recycles apo-Tf back to the cell surface after the release of iron, TfR2 promotes the intracellular deposition of ligand. Tf delivered by TfR2 does not appear to be degraded, which suggests that its delivery to this organelle may be functionally relevant to the storage of iron in overloaded states.

scholarly journals Ear1p and Ssh4p Are New Adaptors of the Ubiquitin Ligase Rsp5p for Cargo Ubiquitylation and Sorting at Multivesicular Bodies

2008 ◽  
Vol 19 (6) ◽  
pp. 2379-2388 ◽  
Author(s):  
Sébastien Léon ◽  
Zoi Erpapazoglou ◽  
Rosine Haguenauer-Tsapis
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Multivesicular Bodies ◽  
Endosomal Sorting ◽  
Frame Fusion ◽  
Vacuolar Targeting

The ubiquitylation of membrane proteins destined for the vacuole/lysosome is essential for their recognition by the endosomal sorting machinery and their internalization into vesicles of multivesicular bodies (MVBs). In yeast, this process requires Rsp5p, an essential ubiquitin ligase of the Nedd4 family. We describe here two redundant proteins, Ear1p and Ssh4p, required for the vacuolar targeting of several cargoes originating from the Golgi or the plasma membrane. Ear1p is an endosomal protein that interacts with Rsp5p through its PPxY motifs, and it is required for the ubiquitylation of selected cargoes before their MVB sorting. In-frame fusion of cargo to ubiquitin overcomes the need for Ear1p/Ssh4p, confirming a role for these proteins in cargo ubiquitylation. Interestingly, Ear1p is itself ubiquitylated by Rsp5p and targeted to the vacuole. Finally, Ear1p overexpression leads to Rsp5p accumulation at endosomes, interfering with some of its functions in trafficking. Therefore, Ear1p/Ssh4p recruit Rsp5p and assist it in its function at MVBs by directing the ubiquitylation of specific cargoes.

Membrane Remodelling During Reticulocyte Maturation

Physiology ◽  
1989 ◽  
Vol 4 (1) ◽  
pp. 37-42
Author(s):  
RM Johnstone ◽  
K Teng
Keyword(s):  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Multivesicular Bodies ◽  
Membrane Processing ◽  
Reticulocyte Maturation ◽  
Immunocytochemical Studies

During maturation, reticulocytes lose many membrane functions, including the transferrin receptor. Immunocytochemical studies reveal that after endocytosis the transferrin receptor (and many other membrane proteins) is packaged into multivesicular bodies. The vesicular contents are externalized by exocytosis. The specificity of such membrane processing underlies the changing properties of the cells' surfaces.

scholarly journals Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach

1983 ◽  
Vol 210 (1) ◽  
pp. 37-47 ◽  
Author(s):  
B T Pan ◽  
R Blostein ◽  
R M Johnstone
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Plasma Membranes ◽  
Plasma Membrane Proteins ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Cell Plasma ◽  
A Subunit

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.

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