scholarly journals A Novel GTPase-activating Protein for ARF6 Directly Interacts with Clathrin and Regulates Clathrin-dependent Endocytosis

2005 ◽  
Vol 16 (4) ◽  
pp. 1617-1628 ◽  
Author(s):  
Kenji Tanabe ◽  
Tetsuo Torii ◽  
Waka Natsume ◽  
Sten Braesch-Andersen ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Heavy Chain ◽  
Membrane Trafficking ◽  
Novel Species ◽  
Small Gtpase ◽  
Transferrin Receptors ◽  
Gtpase Activating Protein ◽  
Multiple Functions ◽  
Adp Ribosylation Factor

ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: Implications in cancer cell biology

2006 ◽  
Vol 97 (9) ◽  
pp. 801-806 ◽  
Author(s):  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Waka Natsume ◽  
Tetsuo Torii ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Membrane Trafficking ◽  
Cell Biology ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

scholarly journals Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.

1997 ◽  
Vol 8 (8) ◽  
pp. 1481-1499 ◽  
Author(s):  
J Mulholland ◽  
A Wesp ◽  
H Riezman ◽  
D Botstein
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Biochemical Analysis ◽  
Small Gtpase ◽  
Secretory Vesicles ◽  
Cytoskeleton Organization ◽  
New Class ◽  
Actin Cytoskeleton Organization ◽  
Vectorial Transport

Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

scholarly journals Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

2012 ◽  
Vol 23 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Andrea L. Marat ◽  
Maria S. Ioannou ◽  
Peter S. McPherson
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Small Gtpase ◽  
Actin Binding ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)–domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1–3/DENND1A–C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

scholarly journals Epstein–Barr virus BDLF2–BMRF2 complex affects cellular morphology

2009 ◽  
Vol 90 (6) ◽  
pp. 1440-1449 ◽  
Author(s):  
Jens-Bernhard Loesing ◽  
Stefano Di Fiore ◽  
Klaus Ritter ◽  
Rainer Fischer ◽  
Michael Kleines
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Actin Cytoskeleton ◽  
Epstein Barr Virus ◽  
Active Form ◽  
Small Gtpase ◽  
Cellular Morphology ◽  
Subcellular Compartment ◽  
Barr Virus ◽  
Epstein Barr

Herpesvirus glycoproteins often form specific heterodimers that can fulfil functions that cannot be carried out by either of the partners acting alone. This study showed that interactions between the Epstein–Barr virus (EBV) multi-spanning transmembrane envelope protein BMRF2 and type II membrane protein BDLF2 influence the way in which these proteins are trafficked in the cell, and hence the subcellular compartment in which they accumulate. When expressed transiently in mammalian cells, BDLF2 accumulated in the endoplasmic reticulum (ER), whereas BMRF2 accumulated in the ER and Golgi apparatus. However, when the two proteins were co-expressed, BDLF2 was transported with BMRF2 to the Golgi apparatus and from there to the plasma membrane, where the proteins co-localized extensively. The distribution of the two proteins at the plasma membrane was reproducibly associated with dramatic changes in cellular morphology, including the formation of enlarged membrane protrusions and cellular processes whose adhesion extremities were organized by the actin cytoskeleton. A dominant-active form of the small GTPase RhoA was epistatic to this morphological phenotype, suggesting that RhoA is a central component of the signalling pathway that reorganizes the cytoskeleton in response to BDLF2–BMRF2. It was concluded that EBV produces a glycoprotein heterodimer that induces changes in cellular morphology through reorganization of the actin cytoskeleton and may facilitate virion spread between cells.

scholarly journals The Small GTPase Arf6 Is Essential for the Tram/Trif Pathway in TLR4 Signaling

2013 ◽  
Vol 289 (3) ◽  
pp. 1364-1376 ◽  
Author(s):  
Tim Van Acker ◽  
Sven Eyckerman ◽  
Lieselotte Vande Walle ◽  
Sarah Gerlo ◽  
Marc Goethals ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Adaptor Proteins ◽  
Small Gtpase ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Tlr4 Signaling ◽  
Mouse Macrophages ◽  
Adp Ribosylation Factor ◽  
Irf3 Activation

Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal−/− mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.

scholarly journals Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11

2003 ◽  
Vol 163 (1) ◽  
pp. 143-154 ◽  
Author(s):  
Blake Riggs ◽  
Wendy Rothwell ◽  
Sarah Mische ◽  
Gilles R.X. Hickson ◽  
Johanne Matheson ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Binding Domain ◽  
Recycling Endosome ◽  
Actin Remodeling ◽  
Membrane Recruitment ◽  
Nuclear Fallout ◽  
Cytoskeleton Remodeling ◽  
Adp Ribosylation Factor ◽  
Cortical Cytoskeleton

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homologue of arfophilin-2, an ADP ribosylation factor effector that binds Rab11 and influences recycling endosome (RE) organization. Here, we show that Nuf is an important component of the RE, and that these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component. GST pull-downs and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation.

scholarly journals GOLPH3 drives cell migration by promoting Golgi reorientation and directional trafficking to the leading edge

2016 ◽  
Vol 27 (24) ◽  
pp. 3828-3840 ◽  
Author(s):  
Mengke Xing ◽  
Marshall C. Peterman ◽  
Robert L. Davis ◽  
Karen Oegema ◽  
Andrew K. Shiau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Leading Edge ◽  
Invasion And Metastasis ◽  
Oncogenic Driver ◽  
Phosphatidylinositol 4 ◽  
Directional Cell Migration ◽  
Insight Into

The mechanism of directional cell migration remains an important problem, with relevance to cancer invasion and metastasis. GOLPH3 is a common oncogenic driver of human cancers, and is the first oncogene that functions at the Golgi in trafficking to the plasma membrane. Overexpression of GOLPH3 is reported to drive enhanced cell migration. Here we show that the phosphatidylinositol-4-phosphate/GOLPH3/myosin 18A/F-actin pathway that is critical for Golgi–to–plasma membrane trafficking is necessary and limiting for directional cell migration. By linking the Golgi to the actin cytoskeleton, GOLPH3 promotes reorientation of the Golgi toward the leading edge. GOLPH3 also promotes reorientation of lysosomes (but not other organelles) toward the leading edge. However, lysosome function is dispensable for migration and the GOLPH3 dependence of lysosome movement is indirect, via GOLPH3’s effect on the Golgi. By driving reorientation of the Golgi to the leading edge and driving forward trafficking, particularly to the leading edge, overexpression of GOLPH3 drives trafficking to the leading edge of the cell, which is functionally important for directional cell migration. Our identification of a novel pathway for Golgi reorientation controlled by GOLPH3 provides new insight into the mechanism of directional cell migration with important implications for understanding GOLPH3’s role in cancer.

scholarly journals ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

2003 ◽  
Vol 162 (4) ◽  
pp. 647-659 ◽  
Author(s):  
Yoshikatsu Aikawa ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Membrane Trafficking ◽  
Cell Types ◽  
Neuroendocrine Cells ◽  
Dense Core Vesicle ◽  
Type I ◽  
Endosomal Membranes ◽  
Phosphatidylinositol 4 ◽  
Adp Ribosylation Factor

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIγ restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIγ with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.

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