Estimation of the amount of internalized ricin that reaches the trans-Golgi network

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

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Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

Journal of Cell Science ◽

10.1242/jcs.112.11.1721 ◽

1999 ◽

Vol 112 (11) ◽

pp. 1721-1732 ◽

Cited By ~ 2

Author(s):

M.J. Francis ◽

E.E. Jones ◽

E.R. Levy ◽

R.L. Martin ◽

S. Ponnambalam ◽

...

Keyword(s):

Plasma Membrane ◽

Golgi Apparatus ◽

Transmembrane Domain ◽

Menkes Disease ◽

Cytoplasmic Domain ◽

Endocytic Pathway ◽

Site Directed Mutagenesis ◽

Trans Golgi Network ◽

C Terminus ◽

Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

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A trans-Golgi network retention signal YQRL fused to ricin A chain significantly enhances its cytotoxicity

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2003.12.037 ◽

2004 ◽

Vol 313 (4) ◽

pp. 1053-1057 ◽

Cited By ~ 3

Author(s):

Jinbiao Zhan ◽

Liping Ge ◽

Jiangen Shen ◽

Keyi Wang ◽

Shu Zheng

Keyword(s):

Ricin A Chain ◽

Trans Golgi Network ◽

A Chain ◽

Golgi Network ◽

Retention Signal ◽

Ricin A

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Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells.

The Journal of Cell Biology ◽

10.1083/jcb.111.3.987 ◽

1990 ◽

Vol 111 (3) ◽

pp. 987-1000 ◽

Cited By ~ 173

Author(s):

A Wandinger-Ness ◽

M K Bennett ◽

C Antony ◽

K Simons

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Protein Delivery ◽

Mdck Cells ◽

Gradient Centrifugation ◽

Equilibrium Density ◽

Phase Partitioning ◽

Trans Golgi Network ◽

Transport Vesicles ◽

Golgi Network

Immunoisolation techniques have led to the purification of apical and basolateral transport vesicles that mediate the delivery of proteins from the trans-Golgi network to the two plasma membrane domains of MDCK cells. We showed previously that these transport vesicles can be formed and released in the presence of ATP from mechanically perforated cells (Bennett, M. K., A. Wandinger-Ness, and K. Simons, 1988. EMBO (Euro. Mol. Biol. Organ.) J. 7:4075-4085). Using virally infected cells, we have monitored the purification of the trans-Golgi derived vesicles by following influenza hemagglutinin or vesicular stomatitis virus (VSV) G protein as apical and basolateral markers, respectively. Equilibrium density gradient centrifugation revealed that hemagglutinin containing vesicles had a slightly lower density than those containing VSV-G protein, indicating that the two fractions were distinct. Antibodies directed against the cytoplasmically exposed domains of the viral spike glycoproteins permitted the resolution of apical and basolateral vesicle fractions. The immunoisolated vesicles contained a subset of the proteins present in the starting fraction. Many of the proteins were sialylated as expected for proteins existing the trans-Golgi network. The two populations of vesicles contained a number of proteins in common, as well as components which were enriched up to 38-fold in one fraction relative to the other. Among the unique components, a number of transmembrane proteins could be identified using Triton X-114 phase partitioning. This work provides evidence that two distinct classes of vesicles are responsible for apical and basolateral protein delivery. Common protein components are suggested to be involved in vesicle budding and fusion steps, while unique components may be required for specific recognition events such as those involved in protein sorting and vesicle targeting.

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Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.9.3.685 ◽

1998 ◽

Vol 9 (3) ◽

pp. 685-699 ◽

Cited By ~ 56

Author(s):

Kent K. Grindstaff ◽

Robert L. Bacallao ◽

W. James Nelson

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Golgi Complex ◽

Apical Membrane ◽

Basolateral Membrane ◽

Membrane Domains ◽

Trans Golgi Network ◽

G Cells ◽

Polarized Epithelial Cells ◽

Golgi Network

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25–50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) ∼40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

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Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1385 ◽

2015 ◽

Vol 26 (24) ◽

pp. 4401-4411 ◽

Cited By ~ 7

Author(s):

Glen A. Farr ◽

Michael Hull ◽

Emily H. Stoops ◽

Rosalie Bateson ◽

Michael J. Caplan

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Cell Surface ◽

Golgi Complex ◽

Plasma Membranes ◽

Secretory Pathway ◽

Trans Golgi Network ◽

Polarized Epithelial Cells ◽

Golgi Network ◽

E Cadherin

Recent evidence indicates that newly synthesized membrane proteins that share the same distributions in the plasma membranes of polarized epithelial cells can pursue a variety of distinct trafficking routes as they travel from the Golgi complex to their common destination at the cell surface. In most polarized epithelial cells, both the Na,K-ATPase and E-cadherin are localized to the basolateral domains of the plasma membrane. To examine the itineraries pursued by newly synthesized Na,K-ATPase and E-cadherin in polarized MDCK epithelial cells, we used the SNAP and CLIP labeling systems to fluorescently tag temporally defined cohorts of these proteins and observe their behaviors simultaneously as they traverse the secretory pathway. These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase. Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19°C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network. Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking.

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Faculty Opinions recommendation of Interaction of neuronal calcium sensor-1 and ADP-ribosylation factor 1 allows bidirectional control of phosphatidylinositol 4-kinase beta and trans-Golgi network-plasma membrane traffic.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1022716.260676 ◽

2004 ◽

Author(s):

Antonella De Matteis

Keyword(s):

Plasma Membrane ◽

Membrane Traffic ◽

Calcium Sensor ◽

Neuronal Calcium Sensor ◽

Adp Ribosylation ◽

Trans Golgi Network ◽

Phosphatidylinositol 4 ◽

Golgi Network ◽

Adp Ribosylation Factor

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Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

Molecular Biology of the Cell ◽

10.1091/mbc.12.6.1623 ◽

2001 ◽

Vol 12 (6) ◽

pp. 1623-1631 ◽

Cited By ~ 60

Author(s):

Jack Rohrer ◽

Rosalind Kornfeld

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Lysosomal Hydrolases ◽

Intracellular Location ◽

Trans Golgi Network ◽

Cytosolic Domain ◽

Cytosolic Domains ◽

Mannose 6 Phosphate ◽

Green Fluorescent ◽

Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by “uncovering” enzyme (UCE), which removes a coveringN-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: 488YHPL and C-terminal 511NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.

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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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