Low cytoplasmic pH inhibits endocytosis and transport from the trans-Golgi network to the cell surface.

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.

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Phosphorylation of the cation-independent mannose 6-phosphate receptor is closely associated with its exit from the trans-Golgi network.

The Journal of Cell Biology ◽

10.1083/jcb.120.1.67 ◽

1993 ◽

Vol 120 (1) ◽

pp. 67-75 ◽

Cited By ~ 59

Author(s):

S Méresse ◽

B Hoflack

Keyword(s):

Cell Surface ◽

Specific Inhibitor ◽

Cytoplasmic Domain ◽

Single Step ◽

Coated Vesicles ◽

Coated Pits ◽

Trans Golgi Network ◽

Mannose 6 Phosphate ◽

Golgi Network

We have previously shown that two serine residues present in two conserved regions of the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) cytoplasmic domain are phosphorylated in vivo (residues 2421 and 2492 of the full length bovine CI-MPR precursor). In this study, we have used CHO cells to investigate the phosphorylation state of these two serines along the different steps of the CI-MPR exocytic and endocytic recycling pathways. Transport and phosphorylation of the CI-MPR in the biosynthetic pathway were examined using deoxymannojirimycin (dMM), a specific inhibitor of the cis-Golgi processing enzyme alpha-mannosidase I which leads to the accumulation of N-linked high mannose oligosaccharides on glycoproteins. Upon removal of dMM, normal processing to complex-type oligosaccharides (galactosylation and then sialylation) occurs on the newly synthesized glycoproteins, including the CI-MPR which could then be purified and analyzed on lectin affinity columns. Phosphorylation of the newly synthesized CI-MPR was concomitant with the sialylation of its oligosaccharides and appeared as a major albeit transient modification. Phosphorylation of the cell surface CI-MPR was examined during its endocytosis as well as its return to the Golgi using antibody tagging and exogalactosylation. The cell surface CI-MPR was not phosphorylated when it entered clathrin-coated pits or when it moved to the early and late endosomes. In contrast, the surface CI-MPR was phosphorylated when it had been resialylated upon its return to the trans-Golgi network. Subcellular fractionation experiments showed that the phosphorylated CI-MPR and the corresponding kinase were found in clathrin-coated vesicles. Collectively, these results indicate that phosphorylation of the two serines in the CI-MPR cytoplasmic domain is associated with a single step of transport of its recycling pathways and occurs when this receptor is in the trans-Golgi network and/or has left this compartment via clathrin-coated vesicles.

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Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

Molecular Biology of the Cell ◽

10.1091/mbc.9.11.3241 ◽

1998 ◽

Vol 9 (11) ◽

pp. 3241-3257 ◽

Cited By ~ 254

Author(s):

Wei Chen ◽

Yan Feng ◽

Dayue Chen ◽

Angela Wandinger-Ness

Keyword(s):

Cell Surface ◽

G Protein ◽

Selective Inhibition ◽

Significant Inhibition ◽

Rab Proteins ◽

Wild Type ◽

Trans Golgi Network ◽

Associated Function ◽

Golgi Network ◽

Gdp Dissociation Inhibitor

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum–to–Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.

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VAMP4 cycles from the cell surface to the trans-Golgi network via sorting and recycling endosomes

Journal of Cell Science ◽

10.1242/jcs.03387 ◽

2007 ◽

Vol 120 (6) ◽

pp. 1028-1041 ◽

Cited By ~ 42

Author(s):

T. H. T. Tran ◽

Q. Zeng ◽

W. Hong

Keyword(s):

Cell Surface ◽

Recycling Endosomes ◽

Trans Golgi Network ◽

Golgi Network

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GLUT4 Recycles via a trans-Golgi Network (TGN) Subdomain Enriched in Syntaxins 6 and 16 But Not TGN38: Involvement of an Acidic Targeting Motif

Molecular Biology of the Cell ◽

10.1091/mbc.e02-06-0315 ◽

2003 ◽

Vol 14 (3) ◽

pp. 973-986 ◽

Cited By ~ 157

Author(s):

Annette M. Shewan ◽

Ellen M. van Dam ◽

Sally Martin ◽

Tang Bor Luen ◽

Wanjin Hong ◽

...

Keyword(s):

Cell Surface ◽

Glucose Transporter ◽

Adipocyte Differentiation ◽

Attachment Protein ◽

Trans Golgi Network ◽

Sensitive Factor ◽

Protein Receptors ◽

Glucose Transporter Glut4 ◽

Golgi Network ◽

Endosomal System

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-solubleN-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of thetrans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.

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The steady state distribution of humTGN46 is not significantly altered in cells defective in clathrin-mediated endocytosis

Journal of Cell Science ◽

10.1242/jcs.111.23.3451 ◽

1998 ◽

Vol 111 (23) ◽

pp. 3451-3458 ◽

Cited By ~ 3

Author(s):

G. Banting ◽

R. Maile ◽

E.P. Roquemore

Keyword(s):

Steady State ◽

Cell Surface ◽

Dominant Negative ◽

Type I ◽

Steady State Distribution ◽

State Distribution ◽

Trans Golgi Network ◽

Defective Mutant ◽

Dynamin I ◽

Golgi Network

It has been shown previously that whilst the rat type I integral membrane protein TGN38 (ratTGN38) is predominantly localised to the trans-Golgi network this protein does reach the cell surface from where it is internalised and delivered back to the trans-Golgi network. This protein thus provides a suitable tool for the investigation of trafficking pathways between the trans-Golgi network and the cell surface and back again. The human orthologue of ratTGN38, humTGN46, behaves in a similar fashion. These proteins are internalised from the cell surface via clathrin mediated endocytosis, a process which is dependent upon the GTPase activity of dynamin. We thus reasoned that humTGN46 would accumulate at the surface of cells rendered defective in clathrin mediated endocytosis by virtue of the fact that they express a GTPase defective mutant of dynamin I. It did not. In fact, expression of a dominant negative GTPase defective mutant of dynamin I had no detectable effect on the steady state distribution of humTGN46. One explanation for this observation is that humTGN46 does not travel directly to the cell surface from the trans-Golgi network. Further studies on cells expressing the dominant negative GTPase defective mutant of dynamin I indicate that the major recycling pathway for humTGN46 is in fact between the trans-Golgi network and the early endosome.

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Polarization of myelinating Schwann cell surface membranes: role of microtubules and the trans-Golgi network

Journal of Neuroscience ◽

10.1523/jneurosci.15-03-01797.1995 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1797-1807 ◽

Cited By ~ 42

Author(s):

BD Trapp ◽

GJ Kidd ◽

P Hauer ◽

E Mulrenin ◽

CA Haney ◽

...

Keyword(s):

Schwann Cell ◽

Cell Surface ◽

Trans Golgi Network ◽

Myelinating Schwann Cell ◽

Golgi Network

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Acidification of the cytosol inhibits endocytosis from coated pits.

The Journal of Cell Biology ◽

10.1083/jcb.105.2.679 ◽

1987 ◽

Vol 105 (2) ◽

pp. 679-689 ◽

Cited By ~ 219

Author(s):

K Sandvig ◽

S Olsnes ◽

O W Petersen ◽

B van Deurs

Keyword(s):

Cell Surface ◽

Lucifer Yellow ◽

Fluid Phase ◽

Endocytic Pathway ◽

Electron Microscopic ◽

Coated Pits ◽

Internal Ph ◽

Microscopic Studies ◽

Epidermal Growth ◽

In Cells

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transferrin receptors were present in approximately 75% of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endocytic uptake of transferrin at internal pH less than 6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.

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Adaptor and Clathrin Exchange at the Plasma Membrane andtrans-Golgi Network

Molecular Biology of the Cell ◽

10.1091/mbc.e02-06-0353 ◽

2003 ◽

Vol 14 (2) ◽

pp. 516-528 ◽

Cited By ~ 66

Author(s):

Xufeng Wu ◽

Xiaohong Zhao ◽

Rosa Puertollano ◽

Juan S. Bonifacino ◽

Evan Eisenberg ◽

...

Keyword(s):

Plasma Membrane ◽

Fluorescence Recovery After Photobleaching ◽

Fluorescence Recovery ◽

Coated Vesicle ◽

Coated Pits ◽

Trans Golgi Network ◽

Rapid Exchange ◽

Dynamic Structures ◽

Golgi Network ◽

Network Exchange

We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at thetrans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K+ depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.

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Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0365 ◽

2004 ◽

Vol 15 (2) ◽

pp. 861-869 ◽

Cited By ~ 31

Author(s):

Yaya Lefkir ◽

Marilyne Malbouyres ◽

Daniel Gotthardt ◽

Adrian Ozinsky ◽

Sophie Cornillon ◽

...

Keyword(s):

Early Stage ◽

Fluid Phase ◽

Small Particles ◽

Medium Chain ◽

Murine Macrophages ◽

Fluid Phase Endocytosis ◽

Phagocytic Cups ◽

Golgi Network ◽

Mutant Cells

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1-cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1-cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1-cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.

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