The N-terminal metal-binding site 2 of the Wilson's Disease Protein plays a key role in the transfer of copper from Atox1

The Wilson's disease protein (WNDP) is a copper-transporting ATPase regulating distribution of copper in the liver. Mutations in WNDP lead to a severe metabolic disorder, Wilson's disease. The function of WNDP depends on Atox1, a cytosolic metallochaperone that delivers copper to WNDP. We demonstrate that the metal-binding site 2 (MBS2) in the N-terminal domain of WNDP (N-WNDP) plays an important role in this process. The transfer of one copper from Atox1 to N-WNDP results in selective protection of the metal-coordinating cysteines in MBS2 against labeling with a cysteine-directed probe. Such selectivity is not observed when free copper is added to N-WNDP. Similarly, site-directed mutagenesis of MBS2 eliminates stimulation of the catalytic activity of WNDP by the copper-Atox1 complex but not by free copper. The Atox1 preference toward MBS2 is likely due to specific protein-protein interactions and is not due to unique surface exposure of the metal-coordinating residues or higher copper binding affinity of MBS2 compared with other sites. Competition experiments using a copper chelator revealed that MBS2 retained copper much better than Atox1, and this may facilitate the metal transfer process. X-ray absorption spectroscopy of the isolated recombinant MBS2 demonstrated that this sub-domain coordinates copper with a linear biscysteinate geometry, very similar to that of Atox1. Therefore, non-coordinating residues in the vicinity of the metal-binding sites are responsible for the difference in the copper binding properties of MBS2 and Atox1. The intramolecular changes that accompany transfer of a single copper to N-WNDP are discussed.     

Human copper-transporting ATPase ATP7B (the Wilson's disease protein): biochemical properties and regulation

Wilson's disease protein (WNDP) is a product of a gene ATP7B that is mutated in patients with Wilson's disease, a severe genetic disorder with hepatic and neurological manifestations caused by accumulation of copper in the liver and brain. In a cell, WNDP transports copper across various cell membranes using energy of ATP-hydrolysis. Copper regulates WNDP at several levels, modulating its catalytic activity, posttranslational modification, and intracellular localization. This review summarizes recent studies on enzymatic function and copper-dependent regulation of WNDP. Specifically, we describe the molecular architecture and major biochemical properties of WNDP, discuss advantages of the recently developed functional expression of WNDP in insect cells, and summarize the results of the ligand-binding studies and molecular modeling experiments for the ATP-binding domain of WNDP. In addition, we speculate on how copper binding may regulate the activity and intracellular distribution of WNDP, and what role the human copper chaperone Atox1 may play in these processes.     

The role of the invariant His-1069 in folding and function of the Wilson's disease protein,the human copper-transporting ATPase ATP7B

The copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in ATP7B lead to Wilson's disease, a severe disorder with neurological and hepatic manifestations. One of the most common disease mutations, a H1069Q substitution, causes intracellular mislocalization of ATP7B (the Wilson's disease protein, WNDP). His-1069 is located in the nucleotide-binding domain of WNDP and is conserved in all copper-transporting ATPases from bacteria to mammals; however, the specific role of this His in the structure and function of WNDP remains unclear. We demonstrate that substitution of His-1069 for Gln, Ala, or Cys does not significantly alter the folding of the WNDP nucleotide-binding domain or the proteolytic resistance of the full-length WNDP. In contrast, the function of WNDP is markedly affected by the mutations. The ability to form an acylphosphate intermediate in the presence of ATP is entirely lost in all three mutants, suggesting that His-1069 is important for ATP-dependent phosphorylation. Other steps of the WNDP enzymatic cycle are less dependent on His-1069. The H1069C mutant shows normal phosphorylation in the presence of inorganic phosphate; it binds an ATP analogue, beta,gamma-imidoadenosine 5'-triphosphate (AMP-PNP), and copper and undergoes nucleotide-dependent conformational transitions similar to those of the wild-type WNDP. Although binding of AMP-PNP is not disrupted by the mutation, the apparent affinity for the nucleotide is decreased by 4-fold. We conclude that His-1069 is responsible for proper orientation of ATP in the catalytic site of WNDP prior to ATP hydrolysis.     

Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.     

Copper specifically regulates intracellular phosphorylation of the Wilson's disease protein, a human copper-transporting ATPase

Copper is a trace element essential for normal cell homeostasis. The major physiological role of copper is to serve as a cofactor to a number of key metabolic enzymes. In humans, genetic defects of copper distribution, such as Wilson's disease, lead to severe pathologies, including neurodegeneration, liver lesions, and behavior abnormalities. Here, we demonstrate that, in addition to its role as a cofactor, copper can regulate important post-translational events such as protein phosphorylation. Specifically, in human cells copper modulates phosphorylation of a key copper transporter, the Wilson's disease protein (WNDP). Copper-induced phosphorylation of WNDP is rapid, specific, and reversible and correlates with the intracellular location of this copper transporter. WNDP is found to have at least two phosphorylation sites, a basal phosphorylation site and a site modified in response to increased copper concentration. Comparative analysis of WNDP, the WNDP pineal isoform, and WNDP C-terminal truncation mutants revealed that the basal phosphorylation site is located in the C-terminal Ser(796)-Tyr(1384) region of WNDP. The copper-induced phosphorylation appears to require the presence of the functional N-terminal domain of this protein. The novel physiological role of copper as a modulator of protein phosphorylation could be central to understanding how copper transport is regulated in mammalian cells.     

Metallochaperone Atox1 transfers copper to the NH2-terminal domain of the Wilson's disease protein and regulates its catalytic activity

Copper is essential for the growth and development of mammalian cells. The key role in the intracellular distribution of copper belongs to the recently discovered family of metallochaperones and to copper-transporting P-type ATPases. The mutations in the ATPase ATP7B, the Wilson's disease protein (WNDP), lead to intracellular accumulation of copper and severe hepatic and neurological abnormalities. Several of these mutations were shown to disrupt the protein-protein interactions between WNDP and the metallochaperone Atox1, suggesting that these interactions are important for normal copper homeostasis. To understand the functional consequences of the Atox1-WNDP interaction at the molecular level, we produced recombinant Atox1 and characterized its effects on WNDP. We demonstrate that Atox1 transfers copper to the purified amino-terminal domain of WNDP (N-WNDP) in a dose-dependent and saturable manner. A maximum of six copper atoms can be transferred to N-WNDP by the chaperone. Furthermore, the incubation of copper Atox1 with the full-length WNDP leads to the stimulation of the WNDP catalytic activity, providing strong evidence for the direct effect of Atox1 on the function of this transporter. Our data also suggest that Atox1 can regulate the copper occupancy of WNDP. The incubation with apo-Atox1 results in the removal of copper from the metalated N-WNDP and apparent down-regulation of WNDP activity. Interestingly, at least one copper atom remains tightly bound to N-WNDP even in the presence of excess apo-Atox1. We suggest that this incomplete reversibility reflects the functional non-equivalency of the metal-binding sites in WNDP and speculate about the intracellular consequences of the reversible Atox1-mediated copper transfer.     

Copper-induced trafficking of the Cu-ATPases: A key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (ccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement ccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.     

The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner

Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010)-Lys(1325) segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed.     

Functional properties of the copper-transporting ATPase ATP7B (the Wilson's disease protein) expressed in insect cells.

Copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in the ATP7B gene lead to copper accumulation in a number of tissues and to a severe multisystem disorder, known as Wilson's disease. Primary sequence analysis suggests that the copper-transporting ATPase ATP7B or the Wilson's disease protein (WNDP) belongs to the large family of cation-transporting P-type ATPases, however, the detailed characterization of its enzymatic properties has been lacking. Here, we developed a baculovirus-mediated expression system for WNDP, which permits direct and quantitative analysis of catalytic properties of this protein. Using this system, we provide experimental evidence that WNDP has functional properties characteristic of a P-type ATPase. It forms a phosphorylated intermediate, which is sensitive to hydroxylamine, basic pH, and treatments with ATP or ADP. ATP stimulates phosphorylation with an apparent K(m) of 0.95 +/- 0.25 microm; ADP promotes dephosphorylation with an apparent K(m) of 3.2 +/- 0.7 microm. Replacement of Asp(1027) with Ala in a conserved sequence motif DKTG abolishes phosphorylation in agreement with the proposed role of this residue as an acceptor of phosphate during the catalytic cycle. Catalytic phosphorylation of WNDP is inhibited by the copper chelator bathocuproine; copper reactivates the bathocuproine-treated WNDP in a specific and cooperative fashion confirming that copper is required for formation of the acylphosphate intermediate. These studies establish the key catalytic properties of the ATP7B copper-transporting ATPase and provide a foundation for quantitative analysis of its function in normal and diseased cells.     

The Wilson's disease protein expressed in Sf9 cells is phosphorylated

The Wilson's disease protein (WNDP), a copper transporter, is a crucial mediator of copper homoeostasis in mammalian cells. We recently found that changes in copper concentration regulate the phosphorylation level of WNDP. WNDP phosphorylation was observed in several mammalian cell lines, suggesting that a common phosphorylation pathway exists in these cells. Here we demonstrate that WNDP expressed in Sf9 insect cells is also phosphorylated, as evidenced by metabolic labelling of these cells with [(32)P]P(i). Because the baculovirus system allows us to generate large amounts of protein, we are using this expression method to isolate WNDP and map the sites of WNDP phosphorylation. The identification of phosphorylation sites is the first step towards understanding the physiological role of WNDP phosphorylation.     

The regulation of catalytic activity of the menkes copper-translocating P-type ATPase. Role of high affinity copper-binding sites

The Menkes protein is a transmembrane copper translocating P-type ATPase. Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency. The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood. We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation. We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model. Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity.     

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.     

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.     

Communication between the N and C Termini Is Required for Copper-stimulated Ser/Thr Phosphorylation of Cu(I)-ATPase (ATP7B)

The Wilson disease protein ATP7B exhibits copper-dependent trafficking. In high copper, ATP7B exits the trans-Golgi network and moves to the apical domain of hepatocytes where it facilitates elimination of excess copper through the bile. Copper levels also affect ATP7B phosphorylation. ATP7B is basally phosphorylated in low copper and becomes more phosphorylated (“hyperphosphorylated”) in elevated copper. The functional significance of hyperphosphorylation remains unclear. We showed that hyperphosphorylation occurs even when ATP7B is restricted to the trans-Golgi network. We performed comprehensive phosphoproteomics of ATP7B in low versus high copper, which revealed that 24 Ser/Thr residues in ATP7B could be phosphorylated, and only four of these were copper-responsive. Most of the phosphorylated sites were found in the N- and C-terminal cytoplasmic domains. Using truncation and mutagenesis, we showed that inactivation or elimination of all six N-terminal metal binding domains did not block copper-dependent, reversible, apical trafficking but did block hyperphosphorylation in hepatic cells. We showed that nine of 15 Ser/Thr residues in the C-terminal domain were phosphorylated. Inactivation of 13 C-terminal phosphorylation sites reduced basal phosphorylation and eliminated hyperphosphorylation, suggesting that copper binding at the N terminus propagates to the ATP7B C-terminal region. C-terminal mutants with either inactivating or phosphomimetic substitutions showed little effect upon copper-stimulated trafficking, indicating that trafficking does not depend on phosphorylation at these sites. Thus, our studies revealed that copper-dependent conformational changes in the N-terminal region lead to hyperphosphorylation at C-terminal sites, which seem not to affect trafficking and may instead fine-tune copper sequestration.     

Characterization of the interaction between the Wilson and Menkes disease proteins and the cytoplasmic copper chaperone, HAH1p.

Wilson disease (WD) and Menkes disease (MNK) are inherited disorders of copper metabolism. The genes that mutate to give rise to these disorders encode highly homologous copper transporting ATPases. We use yeast and mammalian two-hybrid systems, along with an in vitro assay to demonstrate a specific, copper-dependent interaction between the six metal-binding domains of the WD and MNK ATPases and the cytoplasmic copper chaperone HAH1. We demonstrate that several metal-binding domains interact independently or in combination with HAH1p, although notably domains five and six of WDp do not. Alteration of either the Met or Thr residue of the HAH1p MTCXXC motif has no observable effect on the copper-dependent interaction, whereas alteration of either of the two Cys residues abolishes the interaction. Mutation of any one of the HAH1p C-terminal Lys residues (Lys(56), Lys(57), or Lys(60)) to Gly does not affect the interaction, although deletion of the 15 C-terminal residues abolishes the interaction. We show that apo-HAH1p can bind in vitro to copper-loaded WDp, suggesting reversibility of copper transfer from HAH1p to WD/MNKp. The in vitro HAH1/WDp interaction is metalospecific; HAH1 preincubated with Cu(2+) or Hg(+) but not with Zn(2+), Cd(2+), Co(2+), Ni(3+), Fe(3+), or Cr(3+) interacted with WDp. Finally, we model the protein-protein interaction and present a theoretical representation of the HAH1p.Cu.WD/MNKp complex.     

The distinct functional properties of the nucleotide-binding domain of ATP7B, the human copper-transporting ATPase: analysis of the Wilson disease mutations E1064A, H1069Q, R1151H, and C1104F

Copper transport by the P(1)-ATPase ATP7B, or Wilson disease protein (WNDP),1 is essential for human metabolism. Perturbation of WNDP function causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the WNDP nucleotide-binding domain (N-domain), where they are predicted to disrupt ATP binding. The mechanism by which the N-domain coordinates ATP is presently unknown, because residues important for nucleotide binding in the better characterized P(2)-ATPases are not conserved within the P(1)-ATPase subfamily. To gain insight into nucleotide binding under normal and disease conditions, we generated the recombinant WNDP N-domain and several WD mutants. Using isothermal titration calorimetry, we demonstrate that the N-domain binds ATP in a Mg(2+)-independent manner with a relatively high affinity of 75 microm, compared with millimolar affinities observed for the P(2)-ATPase N-domains. The WNDP N-domain shows minimal discrimination between ATP, ADP, and AMP, yet discriminates well between ATP and GTP. Similar results were obtained for the N-domain of ATP7A, another P(1)-ATPase. Mutations of the invariant WNDP residues E1064A and H1069Q drastically reduce nucleotide affinities, pointing to the likely role of these residues in nucleotide coordination. In contrast, the R1151H mutant exhibits only a 1.3-fold reduction in affinity for ATP. The C1104F mutation significantly alters protein folding, whereas C1104A does not affect the structure or function of the N-domain. Together, the results directly demonstrate the phenotypic diversity of WD mutations within the N-domain and indicate that the nucleotide-binding properties of the P(1)-ATPases are distinct from those of the P(2)-ATPases.     

The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein

The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper. The experiments described in this report test the hypothesis that the six MBSs are required for this copper-induced trafficking of MNK. Site-directed mutagenesis was used to convert both cysteine residues in the conserved MBS motifs to serines. Mutation of MBS 1, MBS 6, and MBSs 1-3 resulted in a molecule that appeared to relocalize normally with copper, but when MBSs 4-6 or MBSs 1-6 were mutated, MNK remained in the TGN, even when cells were exposed to 300 microM copper. Furthermore, the ability of the MNK variants to relocalize corresponded well with their ability to confer copper resistance. To further define the critical motifs, MBS 5 and MBS 6 were mutated, and these changes abolished the response to copper. The region from amino acid 8 to amino acid 485 was deleted, resulting in mutant MNK that lacked 478 amino acids from the N-terminal region, including the first four MBSs. This truncated molecule responded normally to copper. Moreover, when either one of the remaining MBS 5 and MBS 6 was mutated to GMXSXXS, the resulting proteins were localized to the TGN in low copper and relocalized in response to elevated copper. These experiments demonstrated that the deleted N-terminal region from amino acid 8 to amino acid 485 was not essential for copper-induced trafficking and that one MBS close to the membrane channel of MNK was necessary and sufficient for the copper-induced redistribution.     

Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase

The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.     

Functions of tropomyosin's periodic repeats

Tropomyosin binds along actin filaments and regulates actin-myosin interaction in muscle and nonmuscle cells. Seven periodic amino acid repeats are proposed to correspond to actin binding sites, and the middle periods are important for cooperative activation of actin by myosin. The functional contributions of individual periods were studied in mutants in which periods 2-6 were individually deleted from rat striated muscle alphaalpha-tropomyosin or replaced with a leucine zipper sequence. Unacetylated recombinant tropomyosins were assayed for actin binding, regulation of the actomyosin ATPase with troponin, cooperative myosin S1-induced binding to actin, and thermal stability. Tropomyosin function is relatively insensitive to deletion of period 2, but loss increases as the deletion is shifted toward the C-terminus. Retention of function upon deletion of the periodic repeats is in the order of 2 > 3 approximately 4 approximately 6 > 5. Internal periods are important for specific functions and are not quasiequivalent. Deletion of period 5 (residues 166-207), and especially deletion or replacement of residues 166-188, a constitutively expressed region encoded by exon 5, had severe consequences on actin affinity and cooperative myosin S1-induced binding to actin. Period 6, residues 208-242, part of the troponin binding site, is required for full inhibition of the actomyosin ATPase in the absence of calcium. The effect of the deletion can depend on its context, suggesting that sequence alone is not the only factor important for function. We propose that the local structure and stability, and consequent flexibility, of the coiled coil are major determinants of actin affinity.     

Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a key regulator of copper homeostasis in humans. It has a dual role in supplying copper to essential cuproenzymes in the trans-Golgi network (TGN) and effluxing copper from the cell. These functions are achieved through copper-regulated trafficking of MNK between the TGN and the plasma membrane. However, the exact mechanism(s) which regulate the localisation and biochemical functions of MNK are still unknown. Here we investigated copper-dependent phosphorylation of MNK by a putative protein kinase(s). We found that in the presence of elevated copper there was a substantial increase in phosphorylation of the wild-type MNK in vivo. The majority of copper-dependent phosphorylation was on serine residues in two phosphopeptides. In contrast, there was no up-regulation of phosphorylation of a non-trafficking MNK mutant with mutated cytosolic copper-binding sites. Our findings suggest a potentially important role of kinase-dependent phosphorylation in the regulation of function of the MNK protein.