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

2015 ◽  
Vol 26 (24) ◽  
pp. 4401-4411 ◽  
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.

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

1998 ◽  
Vol 9 (3) ◽  
pp. 685-699 ◽  
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.

scholarly journals Loss of P4 ATPases Drs2p and Dnf3p Disrupts Aminophospholipid Transport and Asymmetry in Yeast Post-Golgi Secretory Vesicles

2006 ◽  
Vol 17 (4) ◽  
pp. 1632-1642 ◽  
Author(s):  
Nele Alder-Baerens ◽  
Quirine Lisman ◽  
Lambert Luong ◽  
Thomas Pomorski ◽  
Joost C.M. Holthuis
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Budding Yeast ◽  
Secretory Vesicles ◽  
Trans Golgi Network ◽  
P Type ◽  
Golgi Network

Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.

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

1988 ◽  
Vol 106 (2) ◽  
pp. 253-267 ◽  
Author(s):  
B van Deurs ◽  
K Sandvig ◽  
OW Petersen ◽  
S Olsnes ◽  
K Simons ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Golgi Complex ◽  
Endocytic Pathway ◽  
Membrane Glycoproteins ◽  
Double Labeling ◽  
Trans Golgi Network ◽  
A Chain ◽  
Golgi Network ◽  
Ricin A

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.

The ΔF508 mutation shortens the biochemical half-life of plasma membrane CFTR in polarized epithelial cells

2001 ◽  
Vol 280 (1) ◽  
pp. C166-C174 ◽  
Author(s):  
Ghanshyam D. Heda ◽  
Mridul Tanwani ◽  
Christopher R. Marino
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Half Life ◽  
Immunoblot Analysis ◽  
Wild Type ◽  
Δf508 Cftr ◽  
Polarized Epithelial Cells ◽  
Physical And Chemical ◽  
Biochemical Stability

Although the biosynthetic arrest of the ΔF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of ΔF508 and wild-type CFTR at the cell surface of transfected LLC-PK1 cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane ΔF508 CFTR to be ∼4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the ΔF508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the ΔF508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation.

scholarly journals Sequences within the Cytoplasmic Domain of Gp180/Carboxypeptidase D Mediate Localization to the Trans-Golgi Network

1999 ◽  
Vol 10 (1) ◽  
pp. 35-46 ◽  
Author(s):  
Francis J. Eng ◽  
Oleg Varlamov ◽  
Lloyd D. Fricker
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Point Mutations ◽  
Cell Surface Receptor ◽  
Type I ◽  
Multiple Sequence ◽  
Trans Golgi Network ◽  
Duck Hepatitis ◽  
Surface Receptor ◽  
Golgi Network

Gp180, a duck protein that was proposed to be a cell surface receptor for duck hepatitis B virus, is the homolog of metallocarboxypeptidase D, a mammalian protein thought to function in the trans-Golgi network (TGN) in the processing of proteins that transit the secretory pathway. Both gp180 and mammalian metallocarboxypeptidase D are type I integral membrane proteins that contain a 58-residue cytosolic C-terminal tail that is highly conserved between duck and rat. To investigate the regions of the gp180 tail involved with TGN retention and intracellular trafficking, gp180 and various deletion and point mutations were expressed in the AtT-20 mouse pituitary corticotroph cell line. Full length gp180 is enriched in the TGN and also cycles to the cell surface. Truncation of the C-terminal 56 residues of the cytosolic tail eliminates the enrichment in the TGN and the retrieval from the cell surface. Truncation of 12–43 residues of the tail reduced retention in the TGN and greatly accelerated the turnover of the protein. In contrast, deletion of the C-terminal 45 residues, which truncates a potential YxxL-like sequence (FxxL), reduced the protein turnover and caused accumulation of the protein on the cell surface. A point mutation of the FxxL sequence to AxxL slowed internalization, showing that this element is important for retrieval from the cell surface. Mutation of a pair of casein kinase II sites within an acidic cluster showed that they are also important for trafficking. The present study demonstrates that multiple sequence elements within the cytoplasmic tail of gp180 participate in TGN localization.

scholarly journals Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

2008 ◽  
Vol 19 (5) ◽  
pp. 2059-2068 ◽  
Author(s):  
Lauren Henry ◽  
David R. Sheff
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Recycling Endosome ◽  
Recycling Pathway ◽  
Polarized Epithelial Cells ◽  
Pass Through

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the μ1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: localization to the trans-Golgi network and recycling from the cell surface

1998 ◽  
Vol 111 (7) ◽  
pp. 877-885 ◽  
Author(s):  
O. Varlamov ◽  
L.D. Fricker
Keyword(s):  
Cell Surface ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Full Length ◽  
Recycling Endosomes ◽  
Trans Golgi Network ◽  
Cell Processes ◽  
Golgi Network

Carboxypeptidase D (CPD) is a recently discovered membrane-bound metallocarboxypeptidase that has been proposed to be involved in the post-translational processing of peptides and proteins that transit the secretory pathway. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. Antisera to CPD stain the same intracellular structures as those labeled with furin and wheat germ agglutinin. This distribution is distinct from carboxypeptidase E, which is localized to the secretory vesicles in the cell processes. The perinuclear distribution of CPD is detected even when the AtT-20 cells are treated with brefeldin A for 1–30 minutes, suggesting that CPD is present in the trans-Golgi network (TGN). Although CPD is predominantly found in the TGN, an antiserum to the full length protein is internalized within 15–30 minutes of incubation at 37 degrees C. In contrast, an antiserum raised against the C-terminal region of CPD does not become internalized, suggesting that this domain is cytosolic. The antiserum to the full length CPD is internalized to a structure that co-stains with furin and wheat germ agglutinin, but is distinct from transferrin recycling endosomes. The internalization of CPD is not substantially affected by treatment of the AtT-20 cells with brefeldin A. These data are consistent with the cycling of CPD to the cell surface and back to the TGN. The TGN localization of CPD raises the possibility of a role for this enzyme in the processing of proteins that transit the secretory pathway.

scholarly journals Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

2013 ◽  
Vol 202 (2) ◽  
pp. 241-250 ◽  
Author(s):  
Yuichi Wakana ◽  
Julien Villeneuve ◽  
Josse van Galen ◽  
David Cruz-Garcia ◽  
Mitsuo Tagaya ◽  
...  
Keyword(s):  
Cell Surface ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Protein Transport ◽  
S2 Cells ◽  
Trans Golgi Network ◽  
Drosophila S2 Cells ◽  
Vesicular Stomatitis ◽  
Golgi Network

Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface.

scholarly journals Exomer: a coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast

2006 ◽  
Vol 174 (7) ◽  
pp. 973-983 ◽  
Author(s):  
Chao-Wen Wang ◽  
Susan Hamamoto ◽  
Lelio Orci ◽  
Randy Schekman
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Nucleotide Exchange ◽  
Trans Golgi Network ◽  
Peripheral Proteins ◽  
Golgi Network

Ayeast plasma membrane protein, Chs3p, transits to the mother–bud neck from a reservoir comprising the trans-Golgi network (TGN) and endosomal system. Two TGN/endosomal peripheral proteins, Chs5p and Chs6p, and three Chs6p paralogues form a complex that is required for the TGN to cell surface transport of Chs3p. The role of these peripheral proteins has not been clear, and we now provide evidence that they create a coat complex required for the capture of membrane proteins en route to the cell surface. Sec7p, a Golgi protein required for general membrane traffic and functioning as a nucleotide exchange factor for the guanosine triphosphate (GTP)–binding protein Arf1p, is required to recruit Chs5p to the TGN surface in vivo. Recombinant forms of Chs5p, Chs6p, and the Chs6p paralogues expressed in baculovirus form a complex of approximately 1 MD that binds synthetic liposomes in a reaction requiring acidic phospholipids, Arf1p, and the nonhydrolyzable GTPγS. The complex remains bound to liposomes centrifuged on a sucrose density gradient. Thin section electron microscopy reveals a spiky coat structure on liposomes incubated with the full complex, Arf1p, and GTPγS. We termed the novel coat exomer for its role in exocytosis from the TGN to the cell surface. Unlike other coats (e.g., coat protein complex I, II, and clathrin/adaptor protein complex), the exomer does not form buds or vesicles on liposomes.

scholarly journals Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells

2021 ◽  
Vol 118 (33) ◽  
pp. e2014709118
Author(s):  
Stéphanie Lebreton ◽  
Simona Paladino ◽  
Dandan Liu ◽  
Maria Nitti ◽  
Julia von Blume ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Golgi Complex ◽  
Calcium Binding ◽  
Calcium Concentration ◽  
Apical Surface ◽  
Vital Functions ◽  
Molecular Machinery ◽  
Polarized Epithelial Cells ◽  
Apical Sorting

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

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