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

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.

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Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells

Journal of Cell Science ◽

10.1242/jcs.112.1.21 ◽

1999 ◽

Vol 112 (1) ◽

pp. 21-33 ◽

Cited By ~ 17

Author(s):

D. Toomre ◽

P. Keller ◽

J. White ◽

J.C. Olivo ◽

K. Simons

Keyword(s):

Plasma Membrane ◽

Protein Trafficking ◽

Membrane Traffic ◽

Living Cells ◽

Tubular Structures ◽

Trans Golgi Network ◽

Vesicular Stomatitis ◽

Golgi Network ◽

Color Visualization

The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.

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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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Ent3p and Ent5p Exhibit Cargo-specific Functions in Trafficking Proteins between the Trans-Golgi Network and the Endosomes in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e06-11-1000 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1803-1815 ◽

Cited By ~ 37

Author(s):

Alenka Čopič ◽

Trevor L. Starr ◽

Randy Schekman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Surface ◽

Protein Transport ◽

Adaptor Protein ◽

Additional Function ◽

Trans Golgi Network ◽

Cargo Proteins ◽

Clathrin Adaptor ◽

Dependent Transport ◽

Golgi Network

The phosphoinositide-binding proteins Ent3p and Ent5p are required for protein transport from the trans-Golgi network (TGN) to the vacuole in Saccharomyces cerevisiae. Both proteins interact with the monomeric clathrin adaptor Gga2p, but Ent5p also interacts with the clathrin adaptor protein 1 (AP-1) complex, which facilitates retention of proteins such as Chs3p at the TGN. When both ENT3 and ENT5 are mutated, Chs3p is diverted from an intracellular reservoir to the cell surface. However, Ent3p and Ent5p are not required for the function of AP-1, but rather they seem to act in parallel with AP-1 to retain proteins such as Chs3p at the TGN. They have all the properties of clathrin adaptors, because they can both bind to clathrin and to cargo proteins. Like AP-1, Ent5p binds to Chs3p, whereas Ent3p facilitates the interaction between Gga2p and the endosomal syntaxin Pep12p. Thus, Ent3p has an additional function in Gga-dependent transport to the late endosome. Ent3p also facilitates the association between Gga2p and clathrin; however, Ent5p can partially substitute for this function. We conclude that the clathrin adaptors AP-1, Ent3p, Ent5p, and the Ggas cooperate in different ways to sort proteins between the TGN and the endosomes.

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alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis.

The Journal of Cell Biology ◽

10.1083/jcb.89.1.29 ◽

1981 ◽

Vol 89 (1) ◽

pp. 29-34 ◽

Cited By ~ 71

Author(s):

R B Dickson ◽

M C Willingham ◽

I Pastan

Keyword(s):

Electron Microscope ◽

Cell Surface ◽

Vesicular Stomatitis Virus ◽

Golgi Complex ◽

Colloidal Gold ◽

Microscope Level ◽

Coated Pits ◽

Electron Microscope Level ◽

Receptor Mediated Endocytosis ◽

Vesicular Stomatitis

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.

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Coatomer, but not P200/myosin II, is required for the in vitro formation of trans-Golgi network-derived vesicles containing the envelope glycoprotein of vesicular stomatitis virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.3.1073 ◽

1998 ◽

Vol 95 (3) ◽

pp. 1073-1078 ◽

Cited By ~ 26

Author(s):

J.-P. Simon ◽

T.-H. Shen ◽

I. E. Ivanov ◽

D. Gravotta ◽

T. Morimoto ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Envelope Glycoprotein ◽

Myosin Ii ◽

Trans Golgi Network ◽

Vesicular Stomatitis ◽

Golgi Network

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Dimeric PKD regulates membrane fission to form transport carriers at the TGN

The Journal of Cell Biology ◽

10.1083/jcb.200703166 ◽

2007 ◽

Vol 179 (6) ◽

pp. 1123-1131 ◽

Cited By ~ 90

Author(s):

Carine Bossard ◽

Damien Bresson ◽

Roman S. Polishchuk ◽

Vivek Malhotra

Keyword(s):

Protein Kinase ◽

Cell Surface ◽

Protein Transport ◽

Protein Kinase D ◽

Surface Transport ◽

Trans Golgi Network ◽

Definitive Evidence ◽

Membrane Fission ◽

Golgi Network ◽

Constitutively Active

Protein kinase D (PKD) is recruited to the trans-Golgi network (TGN) through interaction with diacylglycerol (DAG) and is required for the biogenesis of TGN to cell surface transport carriers. We now provide definitive evidence that PKD has a function in membrane fission. PKD depletion by siRNA inhibits trafficking from the TGN, whereas expression of a constitutively active PKD converts TGN into small vesicles. These findings demonstrate that PKD regulates membrane fission and this activity is used to control the size of transport carriers, and to prevent uncontrolled vesiculation of TGN during protein transport.

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Three distinct steps in transport of vesicular stomatitis virus glycoprotein from the ER to the cell surface in vivo with differential sensitivities to GTP gamma S

Journal of Cell Science ◽

10.1242/jcs.111.13.1877 ◽

1998 ◽

Vol 111 (13) ◽

pp. 1877-1888 ◽

Cited By ~ 8

Author(s):

R. Pepperkok ◽

M. Lowe ◽

B. Burke ◽

T.E. Kreis

Keyword(s):

Cell Surface ◽

Vesicular Stomatitis Virus ◽

Golgi Complex ◽

Mammalian Cells ◽

Coat Proteins ◽

Gtp Hydrolysis ◽

Dominant Mutant ◽

Vesicular Stomatitis ◽

Gamma S

Microinjected GTP gamma S revealed three distinct steps in the exocytic transport of the temperature sensitive glycoprotein of vesicular stomatitis virus (ts-O45-G) from the ER to the cell surface in intact Vero cells. While COPII dependent export of ts-O45-G from the ER is blocked in cells injected with recombinant protein of a dominant mutant of SAR1a (SAR1a[H79G]) inhibited in GTP hydrolysis, neither injected GTP gamma S nor antibodies against beta-COP (anti-EAGE) interfere with this transport step significantly. In contrast, transport to the Golgi complex is blocked by 50 microM GTP gamma S, a dominant mutant of ARF1 (ARF1[Q71L]) inhibited in GTP hydrolysis, or microinjected anti-EAGE, but injected Sar1a[H79G]p has no effect. Microinjection of GTP gamma S or expression of ARF[Q71L] rapidly induces accumulation of COPI coated vesicular structures lacking ts-O45-G. Finally, transport of ts-O45-G from the trans-Golgi network (TGN) to the cell surface is inhibited only by high concentrations of GTP gamma S (500 microM). Interestingly, this step is only partially brefeldin A sensitive, and injected antibodies against beta-COP and p200/myosin II, a TGN membrane associated protein, have no effect. These data provide first strong in vivo evidence for at least three distinct steps in the exocytic pathway of mammalian cells regulated by different sets of GTPases and coat proteins. COPII, but not COPI, is required for ER export of ts-O45-G. COPI plays a role in subsequent transport to the Golgi complex, and a so far unidentified GTP gamma S sensitive coat appears to be involved in transport from the TGN to the cell surface.

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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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