Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase)

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.     

Activation of ADP-ribosylation factor regulates biogenesis of the ATP7A-containing trans-Golgi network compartment and its Cu-induced trafficking

ATP7A (MNK) regulates copper homeostasis by translocating from a compartment localized within the trans-Golgi network to the plasma membrane (PM) in response to increased copper load. The mechanisms that regulate the biogenesis of the MNK compartment and the trafficking of MNK are unclear. Here we show that the architecture of the MNK compartment is linked to the structure of the Golgi ribbon. Depletion of p115 tethering factor, which causes fragmentation of the Golgi ribbon, also disrupts the MNK compartment. In p115-depleted cells, MNK localizes to punctate structures that pattern on Golgi ministacks dispersed throughout the cell. Despite altered localization MNK trafficking still occurs, and MNK relocates from and returns to the fragmented compartment in response to copper. We further show that the biogenesis of the MNK compartment requires activation of ADP-ribosylation factor (Arf)1 GTPase, shown previously to facilitate the biogenesis of the Golgi ribbon. Activation of cellular Arf1 is prevented by 1) expressing an inactive "empty" form of Arf (Arf1/N126I), 2) expressing an inactive form of GBF1 (GBF1/E794K), guanine nucleotide exchange factor for Arf1, or 3) treating cells with brefeldin A, an inhibitor of GBF1 that disrupts MNK into a diffuse pattern. Importantly, preventing Arf activation inhibits copper-responsive trafficking of MNK to the PM. Our findings support a model in which active Arf is essential for the generation of the MNK compartment and for copper-responsive trafficking of MNK from there to the PM. Our findings provide an exciting foundation for identifying Arf1 effectors that facilitate the biogenesis of the MNK compartment and MNK traffic.     

Trafficking of the copper-ATPases, ATP7A and ATP7B: role in copper homeostasis

Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.     

Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.     

Arabidopsis TRAPPII is functionally linked to Rab-A,but not Rab-D in polar protein trafficking in trans-Golgi network

The trans-Golgi network (TGN) in plant cells is an independent organelle, displaying rapid association and dissociation with Golgi bodies. In plant cells, the TGN is the site where secretory and endocytic membrane trafficking meet. Cell wall components, signaling molecules and auxin transporters have been found to undergo intracellular trafficking around the TGN. However, how different trafficking pathways are regulated and how different cargoes are sorted in the TGN is poorly defined in plant cells. Using a combined approach of genetic and in vivo imaging, we recently demonstrated that Arabidopsis TRAPPII acts in the TGN and is required for polar targeting of PIN2, but not PIN1, auxin efflux carrier in root tip cells. Here, we report that, TRAPPII in Arabidopsis is required for polar distribution of AUX1, an auxin influx carrier in protophloem cells and epidermal cells of Arabidopsis root tips. In yeast cells, TRAPPII serves as a guanine-nucleotide exchange factor (GEF) for Ypt1 and Ypt31/32 in late Golgi trafficking, while in mammalian cells, TRAPPII acts as a GEF for Rab1 (homolog of yeast Ypt1) in early Golgi trafficking. We show here that TRAPPII in Arabidopsis is functionally linked to Rab-A proteins, homologs of yeast Ypt31/32, but not Rab-D proteins, homologs of yeast Ypt1 and animal Rab1 proteins.     

Oleate-induced aggregation of LC3 at the trans-Golgi network is linked to a protein trafficking blockade

Oleate, the most abundant endogenous and dietary cis-unsaturated fatty acid, has the atypical property to cause the redistribution of microtubule-associated proteins 1A/1B light chain 3B (referred to as LC3) to the trans-Golgi network (TGN), as shown here. A genome-wide screen identified multiple, mostly Golgi transport-related genes specifically involved in the oleate-induced relocation of LC3 to the Golgi apparatus. Follow-up analyses revealed that oleate also caused the retention of secreted proteins in the TGN, as determined in two assays in which the secretion of proteins was synchronized, (i) an assay involving a thermosensitive vesicular stomatitis virus G (VSVG) protein that is retained in the endoplasmic reticulum (ER) until the temperature is lowered, and (ii) an isothermic assay involving the reversible retention of the protein of interest in the ER lumen and that was used both in vitro and in vivo. A pharmacological screen searching for agents that induce LC3 aggregation at the Golgi apparatus led to the identification of “oleate mimetics” that share the capacity to block conventional protein secretion. In conclusion, oleate represents a class of molecules that act on the Golgi apparatus to cause the recruitment of LC3 and to stall protein secretion.Subject terms: Autophagy, Proteins     

Copper-dependent interaction of dynactin subunit p62 with the N terminus of ATP7B but not ATP7A

The P-type ATPase affected in Wilson disease, ATP7B, is a key liver protein required to regulate and maintain copper homeostasis. When hepatocytes are exposed to elevated copper levels, ATP7B traffics from the trans-Golgi network toward the biliary canalicular membrane to excrete excess copper into bile. The N-terminal region of ATP7B contains six metal-binding sites (MBS), each with the copper-binding motif MXCXXC. These sites are required for the activity and copper-regulated intracellular redistribution of ATP7B. Two proteins are known to interact with the ATP7B N-terminal region: the copper chaperone ATOX1 that delivers copper to ATP7B, and COMMD1 (MURR1) that is potentially involved in vesicular copper sequestration. To identify additional proteins that interact with ATP7B and hence are involved in copper homeostasis, a yeast two-hybrid approach was employed to screen a human liver cDNA library. The dynactin subunit p62 (dynactin 4; DCTN4) was identified as an interacting partner, and this interaction was confirmed by co-immunoprecipitation from mammalian cells. The dynactin complex binds cargo, such as vesicles and organelles, to cytoplasmic dynein for retrograde microtubule-mediated trafficking and could feasibly be involved in the copper-regulated trafficking of ATP7B. The ATP7B/p62 interaction required copper, the metal-binding CXXC motifs, and the region between MBS 4 and MBS 6 of ATP7B. The p62 subunit did not interact with the related copper ATPase, ATP7A. We propose that the ATP7B interaction with p62 is a key component of the copper-induced trafficking pathway that delivers ATP7B to subapical vesicles of hepatocytes for the removal of excess copper into bile.     

Purification and functional reconstitution of the human Wilson copper ATPase, ATP7B

Wilson disease is a disorder of copper metabolism, due to inherited mutations in the Wilson copper ATPase gene ATP7B. To purify and study the function of the ATPase, the enzyme was truncated by five of the six metal binding domains and endowed with an N-terminal histidine-tag for affinity purification. This construct, delta1-5WNDP, was able to functionally complement a yeast strain defective in its native copper ATPase CCC2. Delta1-5WNDP was purified by Ni-affinity chromatography and reconstituted into proteoliposomes. This allowed, for the first time, the functional study of the Wilson ATPase in a purified, reconstituted system.     

NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation

The cloned epithelial cell-specificNa⁺/H⁺exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor(FGF), phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), andfetal bovine serum (FBS) through a change in maximal velocity of thetransporter. In the present study, we used COOH-terminal truncationmutants to delineate specific domains in the COOH terminus of NHE2 that are responsible for growth factor and/or protein kinase regulation. Five truncation mutants (designated by the amino acid number at thetruncation site) were stably expressed in NHE-deficient PS120 fibroblasts. The effects of PMA, FGF, OA, FBS, and W-13 [aCa²⁺/calmodulin (CaM)inhibitor] were studied. Truncation mutant E2/660, but notE2/573, was stimulated by PMA. OA stimulated E2/573 but not E2/540. FGFstimulated E2/540 but not E2/499. The most truncated mutant, E2/499,was stimulated by FBS. W-13 stimulated the basal activity of thewild-type NHE2. However, W-13 had no effect on E2/755. By monitoringthe emission spectra of dansylated CaM fluorescence, we showed thatdansylated CaM bound directly to a purified fusion protein ofglutathione S-transferase and the last87 amino acids of NHE2 in aCa²⁺-dependent manner, with astoichiometry of 1:1 and a dissociation constant of 300 nM. Our results showed that the COOH terminus of NHE2 is organized intoseparate stimulatory and inhibitory growth factor/protein kinaseregulatory subdomains. This organization of growth factor/proteinkinase regulatory subdomains is very similar to that of NHE3,suggesting that the tertiary structures of the putative COOH termini ofNHE2 and NHE3 are very similar despite the minimal amino acid identityin this part of the two proteins.     

Effects of different sources of copper on Ctr1, ATP7A,ATP7B,MT and DMT1 protein and gene expression in Caco-2 cells

Copper sulfate (CuSO₄), micron copper oxide (micron CuO) and nano copper oxide (nano CuO) at different concentrations were, respectively, added to culture media containing Caco-2 cells and their effects on Ctr1, ATP7A/7B, MT and DMT1 gene expression and protein expression were investigated and compared. The results showed that nano CuO promoted mRNA expression of Ctr1 in Caco-2 cells, and the difference was significant compared with micron CuO and CuSO₄. Nano CuO was more effective in promoting the expression of Ctr1 protein than CuSO₄ and micron CuO at the same concentration. Nano CuO at a concentration of 62.5 μM increased the mRNA expression levels of ATP7A and ATP7B, and the difference was significant compared with CuSO₄. The addition of CuSO₄ and nano CuO to the culture media promoted the expression of ATP7B proteins. CuSO₄ at a concentration of 125 μM increased the mRNA expression level of MT in Caco-2 cells, and the difference was significant compared with nano CuO and micron CuO. Nano CuO at a concentration of 62.5 μM inhibited the mRNA expression of DMT1, and the difference was significant compared with CuSO₄ and micron CuO. Thus, the effects of CuSO₄, micron CuO and nano CuO on the expression of copper transport proteins and the genes encoding these proteins differed considerably. Nano CuO has a different uptake and transport mechanism in Caco-2 cells to those of CuSO₄ and micron CuO.     

Characterization and regulation of constitutive transport intermediates involved in trafficking from the trans-Golgi network

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans-Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans-Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. An in vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200 nm in length. These membrane-enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.     

Comparative features of copper ATPases ATP7A and ATP7B heterologously expressed in COS-1 cells

ATP7A and ATP7B are P-type ATPases required for copper homeostasis and involved in the etiology of Menkes and Wilson diseases. We used heterologous expression of ATP7A or ATP7B in COS-1 cells infected with adenovirus vectors to characterize differential features pertinent to each protein expressed in the same mammalian cell type, rather than to extrinsic factors related to different cells sustaining expression. Electrophoretic analysis of the expressed protein, before and after purification, prior or subsequent to treatment with endoglycosidase, and evidenced by protein or glycoprotein staining as well as Western blotting, indicates that the ATP7A protein is glycosylated while ATP7B is not. This is consistent with the prevalence of glycosylation motifs in the ATP7A sequence, and not in ATP7B. ATP7A and ATP7B undergo copper-dependent phosphorylation by utilization of ATP, forming equal levels of an "alkali labile" phosphoenzyme intermediate that undergoes similar catalytic (P-type ATPase) turnover in both enzymes. In addition, incubation with ATP yields an "alkali stable" phosphoprotein fraction, attributed to phosphorylation of serines. Alkali stable phosphorylation occurs at lower levels in ATP7A, consistent with a different distribution of serines in the amino acid sequence. Immunostaining of COS-1 cells sustaining heterologous expression shows initial association of both ATP7A and ATP7B with Golgi and the trans-Golgi network. However, in the presence of added copper, ATP7A undergoes prevalent association with the plasma membrane while ATP7B exhibits intense trafficking with cytosolic vesicles. Glycosylation of ATP7A and phosphorylation of ATP7B apparently contribute to their different trafficking and membrane association when expressed in the same cell type.     

Clusterin and COMMD1 independently regulate degradation of the mammalian copper ATPases ATP7A and ATP7B

ATP7A and ATP7B are copper-transporting P(1B)-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis.     

Structures of the human Wilson disease copper transporter ATP7B

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Trafficking of human ADAM 12-L: retention in the trans-Golgi network

We have investigated the trafficking of the membrane-anchored form of human ADAM 12 (ADAM 12-L) fused to a green fluorescence protein tag. Subcellular localization of the protein in transiently transfected cells was determined by fluorescence microscopy and trypsin sensitivity. Full-length ADAM 12-L was retained in a perinuclear compartment, which was shown to be the trans-Golgi network. In contrast, ADAM 12-L lacking the cytoplasmic domain reached the cell surface. Based on analysis of deletions and mutations of the cytoplasmic tail of ADAM 12-L, the retention signal is comprised of both the cytoplasmic and transmembrane domains, but not the Src homology 3 domain (SH3) binding sites. These results raise the possibility that a trafficking checkpoint in the trans-Golgi network is one of the cellular mechanisms for regulation of ADAM 12-L function, by allowing a rapid release of ADAM 12-L to the cell surface under specific stimuli.     

Herpes simplex virus type 1 glycoprotein K and the UL20 protein are interdependent for intracellular trafficking and trans-Golgi network localization

Final envelopment of the cytoplasmic herpes simplex virus type 1 (HSV-1) nucleocapsid is thought to occur by budding into trans-Golgi network (TGN)-derived membranes. The highly membrane-associated proteins UL20p and glycoprotein K (gK) are required for cytoplasmic envelopment at the TGN and virion transport from the TGN to extracellular spaces. Furthermore, the UL20 protein is required for intracellular transport and cell surface expression of gK. Independently expressed gK or UL20p via transient expression in Vero cells failed to be transported from the endoplasmic reticulum (ER). Similarly, infection of Vero cells with either gK-null or UL20-null viruses resulted in ER entrapment of UL20p or gK, respectively. In HSV-1 wild-type virus infections and to a lesser extent in transient gK and UL20p coexpression experiments, both gK and UL20p localized to the Golgi apparatus. In wild-type, but not UL20-null, viral infections, gK was readily detected on cell surfaces. In contrast, transiently coexpressed gK and UL20p predominantly localized to the TGN and were not readily detected on cell surfaces. However, TGN-localized gK and UL20p originated from endocytosed gK and UL20p expressed at cell surfaces. Retention of UL20p to the ER through the addition of an ER retention motif forced total ER retention of gK, indicating that transport of gK is absolutely dependent on UL20p transport. In all experiments, gK and UL20p colocalized at intracellular sites, including the ER, Golgi, and TGN. These results are consistent with the hypothesis that gK and UL20p directly interact and that this interaction facilitates their TGN localization, an important prerequisite for cytoplasmic virion envelopment and egress.     

trans-Golgi retention of a plasma membrane protein: mutations in the cytoplasmic domain of the asialoglycoprotein receptor subunit H1 result in trans-Golgi retention

Unlike the wild-type asialoglycoprotein receptor subunit H1 which is transported to the cell surface, endocytosed and recycled, a mutant lacking residues 4-33 of the 40-amino acid cytoplasmic domain was found to be retained intracellularly upon expression in different cell lines. The mutant protein accumulated in the trans-Golgi, as judged from the acquisition of trans-Golgi-specific modifications of the protein and from the immunofluorescence staining pattern. It was localized to juxtanuclear, tubular structures that were also stained by antibodies against galactosyltransferase and gamma-adaptin. The results of further mutagenesis in the cytoplasmic domain indicated that the size rather than the specific sequence of the cytoplasmic domain determines whether H1 is retained in the trans-Golgi or transported to the cell surface. Truncation to less than 17 residues resulted in retention, and extension of a truncated tail by an unrelated sequence restored surface transport. The transmembrane segment of H1 was not sufficient for retention of a reporter molecule and it could be replaced by an artificial apolar sequence without affecting Golgi localization. The cytoplasmic domain thus appears to inhibit interaction(s) of the exoplasmic portion of H1 with trans-Golgi component(s) for example by steric hindrance or by changing the positioning of the protein in the membrane. This mechanism may also be functional in other proteins.     

COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.