ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and a tubulovesicular compartment GDP HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment ARF6-transfected redistributes ARF6 PM stimulates formation actin-rich surface protrusions. Here we show cytochalasin D (CD) inhibited AlF-induced protrusions shifted distribution tubular emanating from juxtanuclear region cells, which resembled where GTP-binding...

There are many pathways of endocytosis at the cell surface that apparently operate same time. With advent new molecular genetic and imaging tools, an understanding different ways by which a may endocytose cargo is increasing leaps bounds. In this review we explore occur in absence clathrin. These referred to as clathrin-independent (CIE). Here primarily focus on those function small scale some have distinct coats (caveolae) others specific coated intermediates. We follow trafficking...

The coatomer is a cytosolic protein complex that reversibly associates with Golgi membranes and implicated in modulating membrane transport. association of beta-COP, component coatomer, enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), nonhydrolyzable analogue GTP, mixture aluminum fluoride ions (Al/F). Here we show the ADP-ribosylation factor (ARF) required for binding beta-COP. Thus, beta-COP contained fraction has been resolved from ARF does not bind to membranes, whereas...

ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between plasma (PM) and a nonclathrin-derived endosomal compartment activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates 4,5-bisphosphate (PIP2). Here, we show PIP2 visualized by expressing fusion protein pleckstrin homology domain from PLCδ green fluorescent (PH-GFP), colocalized with Arf6 at PM on tubular structures. Activation expression its exchange EFA6 stimulated protrusion...

We discuss here the variety of approaches that have been taken to inhibit different forms endocytosis. Typically, both non-specific and specific chemical inhibitors endocytosis are tried in order "classify" entry a new plasma membrane protein into one various types This classification can be confirmed through genetic depletion or overexpression mutants known machinery components. Although some compounds designed selective biochemical assays, we caution investigators alert unintended...

Brefeldin A (BFA) has a profound effect on the structure of Golgi apparatus, causing proteins to redistribute into ER minutes after drug treatment. Here we describe dissociation 110-kD cytoplasmically oriented peripheral membrane protein (Allan, V. J., and T. E. Kreis. 1986. J. Cell Biol. 103:2229-2239) from apparatus as an early event in BFA action, preceding other morphologic changes. In contrast, were not released but followed during The remained widely dispersed throughout cytoplasm...

The binding of cytosolic coat proteins to organelles may regulate membrane structure and traffic. Evidence is presented that a small guanosine triphosphate (GTP)-binding protein, the adenosine diphosphate ribosylation factor (ARF), reversibly associates with Golgi apparatus in an energy, GTP, fungal metabolite brefeldin A (BFA)-sensitive manner similar to, but distinguishable from, 110-kilodalton protein beta-COP. Addition beta gamma subunits G inhibited association both ARF beta-COP...

Clathrin independent endocytosis (CIE) is a form of present in all cells that mediates the entry nutrients, macromolecules and membrane proteins into cells. When compared to clathrin-dependent (CDE), however, much less known about machinery involved forming CIE endosomes. One way distinguish from CDE has been deplete coat such as clathrin or dynamin GTPase, leading block but not CIE. A drawback genetic manipulations depletion important for mediating over period days can have complex indirect...

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While ARF1 protein has been shown vitro mediate interaction cytosolic coat coatomer (COP1) and gamma-adaptin with Golgi complex, functions other have not defined. Here, we show by transient transfection epitope-tagged ARFs, that whereas is localized complex can be affect predictably assembly COP1 membranes cells, ARF6 endosomal/plasma system no effect on these Golgi-associated...

ABSTRACT The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between Rac1, Rho family GTPase, implicated in rearrangements. Endogenous Rac1 colocalized with at plasma on endosome untransfected HeLa primary human fibroblast cells. In transfected cells also colocalized. Cells expressing wild-type or formed actin-containing surface protrusions ruffles, respectively, upon treatment G protein...

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility class I (MHCI), endogenous, integral protein, entered cells through a mechanism followed...

The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor alpha subunit (Tac) was compared with PM internalized via clathrin. MHCI Tac were into endosomes distinct from those containing cargo. At later times, a fraction these membranes observed in Arf6-associated, tubular recycling whereas another acquired early endosomal autoantigen 1 (EEA1) before fusion the "classical"...

To study the effector function of ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6. Previously shown to indirectly activate ARF1 GTPase, aluminum fluoride (AIF) treatment ARF6-transfected resulted in a redistribution both ARF6 and actin discrete sites on plasma membrane, which became increasingly protrusive over time. The effects AIF were reversible, specific wild-type ARF6, resembled cellular protrusions observed expressing...

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on controlled exchange and hydrolysis GTP. We have purified cloned two variants a 130-kDa phosphatidylinositol 4, 5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a ASAP1b. Both contain pleckstrin homology (PH) domain, zinc finger similar to that found another GAP, three ankyrin (ANK) repeats, proline-rich region with...

Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members the ARF family GTPases are essential for many aspects trafficking. To test a role this proteins in Fcgamma receptor-mediated phagocytosis, we utilized fungal metabolite brefeldin A (BFA). The addition 100 microM BFA to subclone RAW 264.7 macrophages disrupted appearance function Golgi apparatus as indicated altered immunofluorescent distribution beta-COP reduced efflux BODIPY...

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays prefers Arf1 over Arf6 as substrate, its localization cells at plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that activated HeLa and COS-7 resulting recruitment on to dynamic PM ruffles. By contrast, was less by than EFA6, canonical GEF. Remarkably, GTP-bound form recruited two proteins could be immunoprecipitated. binding not mediated...