We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH2-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal “ear” domain of γ-adaptin. However, unlike γ-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the γ-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.
The mouse mutants mocha and pearl are deficient in the AP-3 δ and β3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the β3 and μ3 subunits coassemble into a heterodimer, whereas the σ3 subunit remains monomeric. In pearl cells, the δ and σ3 subunits coassemble into a heterodimer, whereas μ3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of β3 and μ3 can interact with each other. Pearl cell lines were generated that express β3A, β3B, a β3Aβ2 chimera, two β3A deletion mutants, and a β3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only β3A, β3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The β3Aβ2 chimera and the β3A short deletion mutant gave partial functional rescue, whereas the β3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of β3 are important for function, but the clathrin binding site is not needed.
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.
The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the γ-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both γ-adaptin and α-adaptin, and γ-synergin, an alternatively spliced protein with an apparent molecular mass of ∼110–190 kD, which only interacts with γ-adaptin. γ-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to the ear domain of γ-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the γ-adaptin binding site. In cells expressing α-adaptin with the γ-adaptin ear, a construct that goes mainly to the plasma membrane, much of the γ-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that γ-synergin links the AP-1 complex to another protein or proteins.
We have recently identified a novel family of proteins, the GGAs. The GGAs consist of four distinct domains and appear to be monomeric. Studies making use of both yeast and mammalian cells indicate that both the GGAs and the AP-1 adaptor complex facilitate trafficking from the T G N to an endosomal compartment. Our current model is that the GGAs are monomeric adaptors, with the N-terminal VHS domain involved in cargo selection, the GAT domain serving to target the GGAs to the TGN, the variable hinge-like domain interacting with clathrin, and the C-terminal gamma-adaptin ear-like domain recruiting accessory proteins onto the membrane. Using GST pulldowns, we have identified two novel potential accessory proteins, p200 and p56, which bind to both GGA ears and gamma ears. p200 is a highly conserved protein, with homologues in Drosophila, C. elegans, and S. cerevisiae. p56 is a protein predicted to have a large amount of coiled coil structure. Antibodies are currently being raised against both proteins for further characterisation.Assembly of SNARE proteins between opposing membranes is thought to be a key event in intracellular membrane fusion. In vitro, complex forming domains of the individual SNARE proteins are largely unstructured. Complex formation is associated with major conformational rearrangements and increased stability. The central domain of the ternary SNARE complex consists of a four-helix bundle, to which synaptobrevin and syntaxin each contribute a single and SNAP-25 two helices. Syntaxin and SNAP-25 can form a ibinaryi complex consisting of two syntaxins and one SNAP-25. This binary complex consists of a four-helix bundle similar to the ternary complex. To investigate the events of the assembly reaction in detail, thermodynamic and kinetic studies were carried out. The ternary SNARE complex unfolds at temperatures above SOT, whereas refolding occurs only below 65°C. A similar hysteresis between ternary complex formation and unfolding was observed in the presence of denaturant. Hence, it is likely that the pathways for ternary SNARE complex formation and unfolding are different. The binary complex is less stable and reversibly unfolds at temperatures around 45°C. Kinetic data imply that successful interaction of syntaxin and SNAP-25 must precede to allow for ternary complex formation. Together, these data suggest that the binary complex is an intermediate for ternary complex formation.Phospholipase D (PLD) activation has been implicated in the regulation of a number of cellular events including cytoskeletal change and regulated secretion. Both PLDl and 2 are regulated by PtdIns(4,5)P2 through ligation of a PtdIns(4,5)P2 specific P H domain. This domain is important in regulating both membrane association and enzyme activity. PLDl is activated by a Rho family protein, an ARF family protein and protein kinase C. BIAcore studies show the regulation to be mediated by direct, independent molecular interaction. In the RBL2H3 mast cell line antigen stimulates the activation of PLD-dependent secretion. In ce...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.