scholarly journals Golgi-localized, γ-Ear-containing, ADP-Ribosylation Factor-binding Proteins: Roles of the Different Domains and Comparison with AP-1 and Clathrin

2001 ◽  
Vol 12 (11) ◽  
pp. 3573-3588 ◽  
Author(s):  
Jennifer Hirst ◽  
Margaret R. Lindsay ◽  
Margaret S. Robinson
Keyword(s):  
Mammalian Cells ◽  
Binding Proteins ◽  
Coated Vesicles ◽  
Factor Binding ◽  
Adp Ribosylation ◽  
Trans Golgi Network ◽  
Vhs Domain ◽  
Vacuolar Protein ◽  
Golgi Network ◽  
Adp Ribosylation Factor

We have previously identified a novel family of proteins called the GGAs (Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding proteins). These proteins consist of an NH2-terminal VHS domain, followed by a GAT domain, a variable domain, and a γ-adaptin ear homology domain. Studies from our own laboratory and others, making use of both yeast and mammals cells, indicate that the GGAs facilitate trafficking from the trans-Golgi network to endosomes. Here we have further investigated the function of the GGAs. We find that GGA-deficient yeast are not only defective in vacuolar protein sorting but they are also impaired in their ability to process α-factor. Using deletion mutants and chimeras, we show that the VHS domain is required for GGA function and that the VHS domain from Vps27p will not substitute for the GGA VHS domain. In contrast, the γ-adaptin ear homology domain contributes to GGA function but is not absolutely required, and full function can be restored by replacing the GGA ear domain with the γ-adaptin ear domain. Deleting the γ-adaptin gene together with the twoGGA genes exacerbates the phenotype in yeast, suggesting that they function on parallel pathways. In mammalian cells, the association of GGAs with the membrane is extremely unstable, which may account for their absence from purified clathrin-coated vesicles. Double- and triple-labeling immunofluorescence experiments indicate that the GGAs and AP-1 are associated with distinct populations of clathrin-coated vesicles budding from the trans-Golgi network. Together with results from other studies, our findings suggest that the GGAs act as monomeric adaptors, with the four domains involved in cargo selection, membrane localization, clathrin binding, and accessory protein recruitment.

scholarly journals Yeast Golgi-localized, γ-Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

2008 ◽  
Vol 19 (11) ◽  
pp. 4826-4836 ◽  
Author(s):  
Mohamed E. Abazeed ◽  
Robert S. Fuller
Keyword(s):  
Binding Proteins ◽  
Adaptor Protein ◽  
Early Endosome ◽  
Dominant Negative ◽  
Factor Binding ◽  
Trans Golgi Network ◽  
Direct Pathway ◽  
Prevacuolar Compartment ◽  
Golgi Network ◽  
Adp Ribosylation Factor

Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome.

scholarly journals Conserved role for Gga proteins in phosphatidylinositol 4-kinase localization to the trans-Golgi network

2017 ◽  
Vol 114 (13) ◽  
pp. 3433-3438 ◽  
Author(s):  
Lydia Daboussi ◽  
Giancarlo Costaguta ◽  
Razmik Ghukasyan ◽  
Gregory S. Payne
Keyword(s):  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Coated Vesicles ◽  
Coat Proteins ◽  
Trans Golgi Network ◽  
Phosphatidylinositol Kinases ◽  
Vhs Domain ◽  
Phosphatidylinositol 4 ◽  
Golgi Network

Phosphoinositides serve as key membrane determinants for assembly of clathrin coat proteins that drive formation of clathrin-coated vesicles. At the trans-Golgi network (TGN), phosphatidylinositol 4-phosphate (PtdIns4P) plays important roles in recruitment of two major clathrin adaptors, Gga (Golgi-localized, gamma-adaptin ear homology, Arf-binding) proteins and the AP-1 (assembly protein-1) complex. The molecular mechanisms that mediate localization of phosphatidylinositol kinases responsible for synthesis of PtdIns4P at the TGN are not well characterized. We identify two motifs in the yeast phosphatidylinositol 4-kinase, Pik1, which are required for binding to the VHS domain of Gga2. Mutations in these motifs that inhibit Gga2–VHS binding resulted in reduced Pik1 localization and delayed accumulation of PtdIns4P and recruitment of AP-1 to the TGN. The Pik1 homolog in mammals, PI4KIIIβ, interacted preferentially with the VHS domain of GGA2 compared with VHS domains of GGA1 and GGA3. Depletion of GGA2, but not GGA1 or GGA3, specifically affected PI4KIIIβ localization. These results reveal a conserved role for Gga proteins in regulating phosphatidylinositol 4-kinase function at the TGN.

scholarly journals ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.

1994 ◽  
Vol 125 (4) ◽  
pp. 721-732 ◽  
Author(s):  
W Nickel ◽  
L A Huber ◽  
R A Kahn ◽  
N Kipper ◽  
A Barthel ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Plasma Membranes ◽  
Vesicular Transport ◽  
Rat Hepatocytes ◽  
Secretory Vesicles ◽  
Isolated Rat Hepatocytes ◽  
Adp Ribosylation ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Adp Ribosylation Factor

Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with [35S]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was approximately 100-200 nm as determined by electron microscopy. A typical coat structure as described for intra-Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14). Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.

scholarly journals A Family of Proteins with γ-Adaptin and Vhs Domains That Facilitate Trafficking between the Trans-Golgi Network and the Vacuole/Lysosome

2000 ◽  
Vol 149 (1) ◽  
pp. 67-80 ◽  
Author(s):  
Jennifer Hirst ◽  
Winnie W.Y. Lui ◽  
Nicholas A. Bright ◽  
Nicholas Totty ◽  
Matthew N.J. Seaman ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Coated Vesicles ◽  
Carboxypeptidase Y ◽  
Golgi Stack ◽  
Trans Golgi Network ◽  
Terminal Domain ◽  
Vhs Domain ◽  
Golgi Network ◽  
Terminal Domains

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.

scholarly journals Role of the Second Cysteine-rich Domain and Pro275 in Protein Kinase D2 Interaction with ADP-Ribosylation Factor 1, Trans-Golgi Network Recruitment, and Protein Transport

2010 ◽  
Vol 21 (6) ◽  
pp. 1011-1022 ◽  
Author(s):  
Ganesh Varma Pusapati ◽  
Denis Krndija ◽  
Milena Armacki ◽  
Götz von Wichert ◽  
Julia von Blume ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Protein Transport ◽  
Small Gtpase ◽  
Adp Ribosylation ◽  
Trans Golgi Network ◽  
Rich Domain ◽  
Golgi Network ◽  
Adp Ribosylation Factor ◽  
Cysteine Rich Domain

Protein kinase D (PKD) isoenzymes regulate the formation of transport carriers from the trans-Golgi network (TGN) that are en route to the plasma membrane. The PKD C1a domain is required for the localization of PKDs at the TGN. However, the precise mechanism of how PKDs are recruited to the TGN is still elusive. Here, we report that ADP-ribosylation factor (ARF1), a small GTPase of the Ras superfamily and a key regulator of secretory traffic, specifically interacts with PKD isoenzymes. ARF1, but not ARF6, binds directly to the second cysteine-rich domain (C1b) of PKD2, and precisely to Pro275 within this domain. Pro275 in PKD2 is not only crucial for the PKD2-ARF1 interaction but also for PKD2 recruitment to and PKD2 function at the TGN, namely, protein transport to the plasma membrane. Our data suggest a novel model in which ARF1 recruits PKD2 to the TGN by binding to Pro275 in its C1b domain followed by anchoring of PKD2 in the TGN membranes via binding of its C1a domain to diacylglycerol. Both processes are critical for PKD2-mediated protein transport.

scholarly journals Vps10p Transport from the trans-Golgi Network to the Endosome Is Mediated by Clathrin-coated Vesicles

2001 ◽  
Vol 12 (2) ◽  
pp. 475-485 ◽  
Author(s):  
Olivier Deloche ◽  
Bonny G. Yeung ◽  
Gregory S. Payne ◽  
Randy Schekman
Keyword(s):  
Large Subunit ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Carboxypeptidase Y ◽  
Yeast Saccharomyces Cerevisiae ◽  
Trans Golgi Network ◽  
Protein Receptor ◽  
Vacuolar Protein ◽  
Golgi Network

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12Δ andvps34Δ, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10CtΔp mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.

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