Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation.
(17/69)
BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell. (+info)
Differential use of myristoyl groups on neuronal calcium sensor proteins as a determinant of spatio-temporal aspects of Ca2+ signal transduction.
(18/69)
The localizations of three members of the neuronal calcium sensor (NCS) family were studied in HeLa cells. Using hippocalcin-EYFP and NCS-1-ECFP, it was found that their localization differed dramatically in resting cells. NCS-1 had a distinct predominantly perinuclear localization (similar to trans-Golgi markers), whereas hippocalcin was present diffusely throughout the cell. Upon the elevation of intracellular Ca(2+), hippocalcin rapidly translocated to the same perinuclear compartment as NCS-1. Another member of the family, neurocalcin delta, also translocated to this region after a rise in Ca(2+) concentration. Permeabilization of transfected cells using digitonin caused loss of hippocalcin and neurocalcin delta in the absence of calcium, but in the presence of 10 microm Ca(2+), both proteins translocated to and were retained in the perinuclear region. NCS-1 localization was unchanged in permeabilized cells regardless of calcium concentration. The localization of NCS-1 was unaffected by mutations in all functional EF hands, indicating that its localization was independent of Ca(2+). A minimal myristoylation motif (hippocalcin-(1-14)) fused to EGFP resulted in similar perinuclear targeting, showing that localization of these proteins is because of the exposure of the myristoyl group. This was confirmed by mutation of the myristoyl motif of NCS-1 and hippocalcin that resulted in both proteins remaining cytosolic, even at elevated Ca(2+) concentration. Dual imaging of hippocalcin-EYFP and cytosolic Ca(2+) concentration in Fura Red-loaded cells demonstrated the kinetics of the Ca(2+)/myristoyl switch in living cells and showed that hippocalcin rapidly translocated with a half-time of approximately 12 s after a short lag period when Ca(2+) was elevated. These results demonstrate that closely related Ca(2+) sensor proteins use their myristoyl groups in distinct ways in vivo in a manner that will determine the time course of Ca(2+) signal transduction. (+info)
Gamma-adaptin interacts directly with Rabaptin-5 through its ear domain.
(19/69)
In yeast two-hybrid screening using gamma1-adaptin, a subunit of the AP-1 adaptor complex of clathrin-coated vesicles derived from the trans-Golgi network (TGN), as bait, we found that it could interact with Rabaptin-5, an effector of Rab5 and Rab4 that regulates membrane docking with endosomes. Further two-hybrid analysis revealed that the interaction occurs between the ear domain of gamma1-adaptin and the COOH-terminal coiled-coil region of Rabaptin-5. Pull down assay with a fusion protein between glutathione S-transferase and the ear domain of gamma1-adaptin and coimmunoprecipitation analysis revealed that the interaction occurs in vitro and in vivo. Immunocytochemical analysis showed that gamma1-adaptin and Rabaptin-5 colocalize to a significant extent on perinuclear structures, probably on recycling endosomes, and are redistributed into the cytoplasm upon treatment with brefeldin A. These results suggest that the gamma1-adaptin-Rabaptin-5 interaction may play a role in membrane trafficking between the TGN and endosomes. (+info)
Retrograde transport of protein toxins under conditions of COPI dysfunction.
(20/69)
Retrograde transport dependent on coat protein I (COPI) was impaired using two different approaches and the effects on the retrograde transport of protein toxins were investigated. One approach was to study ldlF cells that express a temperature-sensitive defect in the epsilon-COP subunit of COPI. The second approach was to treat cells with 1,3-cyclohexanebis(methylamine) (CBM), a drug that interferes with the binding of COPI to Golgi membranes. With both approaches, cells remained sensitive to a variety of protein toxins regardless of whether the toxins contained a KDEL motif. Moreover, cholera toxin, which contains a KDEL sequence, was observed by immunofluorescence microscopy to enter the endoplasmic reticulum of Vero cells in the presence of CBM. These data support published evidence indicating the presence in cells of a COPI- and KDEL receptor-independent pathway of retrograde transport from the Golgi complex to the endoplasmic reticulum. In addition, the results suggest that certain toxins containing a KDEL motif may use either the COPI-dependent or COPI-independent pathway of retrograde transport. (+info)
Gamma-adaptin appendage domain: structure and binding site for Eps15 and gamma-synergin.
(21/69)
The AP1 complex is one of a family of heterotetrameric clathrin-adaptor complexes involved in vesicular trafficking between the Golgi and endosomes. The complex has two large subunits, gamma and beta1, which can be divided into trunk, hinge, and appendage domains. The 1.8 A resolution structure of the gamma appendage is presented. The binding site for the known gamma appendage ligand gamma-synergin is mapped through creation of point mutations designed on the basis of the structure. We also show that Eps15, a protein believed to be involved in vesicle formation at the plasma membrane, is also a ligand of gamma appendage and binds to the same site as gamma-synergin. This observation explains the demonstrated brefeldinA (BFA)-sensitive colocalization of Eps15 and AP1 at the Golgi complex. (+info)
Clint: a novel clathrin-binding ENTH-domain protein at the Golgi.
(22/69)
We have characterized a novel clathrin-binding 68-kDa epsin N-terminal homology domain (ENTH-domain) protein that we name clathrin interacting protein localized in the trans-Golgi region (Clint). It localizes predominantly to the Golgi region of epithelial cells as well as to more peripheral vesicular structures. Clint colocalizes with AP-1 and clathrin only in the perinuclear area. Recombinantly expressed Clint interacts directly with the gamma-appendage domain of AP-1, with the clathrin N-terminal domain through the peptide motif (423)LFDLM, with the gamma-adaptin ear homology domain of Golgi-localizing, gamma-adaptin ear homology domain 2, with the appendage domain of beta2-adaptin and to a lesser extent with the appendage domain of alpha-adaptin. Moreover, the Clint ENTH-domain asssociates with phosphoinositide-containing liposomes. A significant amount of Clint copurifies with rat liver clathrin-coated vesicles. In rat kidney it is preferentially expressed in the apical region of epithelial cells that line the collecting duct. Clathrin and Clint also colocalize in the apical region of enterocytes along the villi of the small intestine. Apart from the ENTH-domain Clint has no similarities with the epsins AP180/CALM or Hip1/1R. A notable feature of Clint is a carboxyl-terminal methionine-rich domain (Met(427)-Met(605)), which contains >17% methionine. Our results suggest that Clint might participate in the formation of clathrin-coated vesicles at the level of the trans-Golgi network and remains associated with the vesicles longer than clathrin and adaptors. (+info)
Rabaptin-5alpha/rabaptin-4 serves as a linker between rab4 and gamma(1)-adaptin in membrane recycling from endosomes.
(23/69)
Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling. (+info)
Binding partners for the COOH-terminal appendage domains of the GGAs and gamma-adaptin.
(24/69)
The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 gamma subunit and the GGAs: gamma-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and gamma-synergin bind to both GGA and gamma appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas gamma-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1-deficient cells, p56 remains membrane-associated whereas gamma-synergin becomes cytosolic. Thus, p56 and gamma-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and gamma appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and gamma appendage domains in cells, we can drive p56 and gamma-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways. (+info)