P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Δ cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.

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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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Soi3p/Rav1p Functions at the Early Endosome to Regulate Endocytic Trafficking to the Vacuole and Localization of Trans-Golgi Network Transmembrane Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e03-10-0755 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3196-3209 ◽

Cited By ~ 27

Author(s):

György Sipos ◽

Jason H. Brickner ◽

E.J. Brace ◽

Linyi Chen ◽

Alain Rambourg ◽

...

Keyword(s):

Vacuolar Atpase ◽

Early Endosome ◽

Trans Golgi Network ◽

Peripheral Protein ◽

Early Endosomes ◽

Localization Signal ◽

Prevacuolar Compartment ◽

Gradient Fractionation ◽

Vacuolar Protein ◽

Golgi Network

SOI3 was identified by a mutation, soi3-1, that suppressed a mutant trans-Golgi network (TGN) localization signal in the Kex2p cytosolic tail. SOI3, identical to RAV1, encodes a protein important for regulated assembly of vacuolar ATPase. Here, we show that Soi3/Rav1p is required for transport between the early endosome and the late endosome/prevacuolar compartment (PVC). By electron microscopy, soi3-1 mutants massively accumulated structures that resembled early endosomes. soi3Δ mutants exhibited a kinetic delay in transfer of the endocytic tracer dye FM4-64, from the 14°C endocytic intermediate to the vacuole. The soi3Δ mutation delayed vacuolar degradation but not internalization of the a-factor receptor Ste3p. By density gradient fractionation, Soi3/Rav1p associated as a peripheral protein with membranes of a density characteristic of early endosomes. The soi3 null mutation markedly reduced the rate of Kex2p transport from the TGN to the PVC but had no effect on vacuolar protein sorting or cycling of Vps10p. These results suggest that assembly of vacuolar ATPase at the early endosome is required for transport of both Ste3p and Kex2p from the early endosome to the PVC and support a model in which cycling through the early endosome is part of the normal itinerary of Kex2p and other TGN-resident proteins.

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The Arf GAP SMAP2 is necessary for organized vesicle budding from the trans-Golgi network and subsequent acrosome formation in spermiogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e13-05-0234 ◽

2013 ◽

Vol 24 (17) ◽

pp. 2633-2644 ◽

Cited By ~ 25

Author(s):

Tomo Funaki ◽

Shunsuke Kon ◽

Kenji Tanabe ◽

Waka Natsume ◽

Sayaka Sato ◽

...

Keyword(s):

Membrane Vesicles ◽

Round Spermatid ◽

Snare Complex ◽

Coated Vesicle ◽

Vesicle Budding ◽

Trans Golgi Network ◽

Protein Receptor ◽

Acrosome Formation ◽

Golgi Network ◽

Coated Membrane

The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.

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SMAP2, a Novel ARF GTPase-activating Protein, Interacts with Clathrin and Clathrin Assembly Protein and Functions on the AP-1–positive Early Endosome/Trans-Golgi Network

Molecular Biology of the Cell ◽

10.1091/mbc.e05-10-0909 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2592-2603 ◽

Cited By ~ 51

Author(s):

Waka Natsume ◽

Kenji Tanabe ◽

Shunsuke Kon ◽

Naomi Yoshida ◽

Toshio Watanabe ◽

...

Keyword(s):

Brefeldin A ◽

Adaptor Proteins ◽

Early Endosome ◽

Dependent Manner ◽

Transferrin Receptors ◽

Gtpase Activating Protein ◽

Trans Golgi Network ◽

Early Endosomes ◽

Golgi Network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.

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The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans-Golgi Network

Molecular Biology of the Cell ◽

10.1091/mbc.e07-06-0622 ◽

2007 ◽

Vol 18 (12) ◽

pp. 4979-4991 ◽

Cited By ~ 62

Author(s):

Zi Zhao Lieu ◽

Merran C. Derby ◽

Rohan D. Teasdale ◽

Charles Hart ◽

Priscilla Gunn ◽

...

Keyword(s):

Shiga Toxin ◽

Retrograde Transport ◽

Transport Pathway ◽

Transport Pathways ◽

Recycling Endosomes ◽

Trans Golgi Network ◽

Early Endosomes ◽

Cargo Proteins ◽

Mannose 6 Phosphate ◽

Golgi Network

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane–TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.

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Protein kinase D2-regulated protein transport at the trans-Golgi network requires its targeting to this compartment by ADP-ribosylation factor 1

Zeitschrift für Gastroenterologie ◽

10.1055/s-0030-1263799 ◽

2010 ◽

Vol 48 (08) ◽

Author(s):

GV Pusapati ◽

G Adler ◽

T Seufferlein

Keyword(s):

Protein Kinase ◽

Protein Transport ◽

Adp Ribosylation ◽

Trans Golgi Network ◽

Golgi Network ◽

Adp Ribosylation Factor

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Faculty Opinions recommendation of Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis.

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

10.3410/f.13353960.14725058 ◽

2011 ◽

Author(s):

Jiri Friml ◽

Steffen Vanneste

Keyword(s):

Early Endosome ◽

Multivesicular Bodies ◽

Trans Golgi Network ◽

Golgi Network

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Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network

The Journal of Cell Biology ◽

10.1083/jcb.200403120 ◽

2004 ◽

Vol 165 (6) ◽

pp. 781-788 ◽

Cited By ~ 91

Author(s):

Sebastien Carreno ◽

Åsa E. Engqvist-Goldstein ◽

Claire X. Zhang ◽

Kent L. McDonald ◽

David G. Drubin

Keyword(s):

Time Lapse ◽

Actin Dynamics ◽

Coated Vesicle ◽

Cell Cortex ◽

Actin Assembly ◽

Trans Golgi Network ◽

Lysosome Biogenesis ◽

Diverse Species ◽

Golgi Network ◽

Golgi Organization

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.

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