ATP7A; MNK - Semantic Scholar

© 1999 Oxford University Press

Human Molecular Genetics, 1999, Vol. 8, No. 11 2107–2115

The Menkes protein (ATP7A; MNK) cycles via the plasma membrane both in basal and elevated extracellular copper using a C-terminal di-leucine endocytic signal Michael J. Petris1,2 and Julian F.B. Mercer2,+ 1The

Murdoch Institute, Royal Children’s Hospital, Parkville 3052, Australia, and 2Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Building L, Deakin University—Burwood Campus, 221 Burwood Highway, Burwood, Victoria 3125, Australia Received June 15, 1999; Revised and Accepted July 30, 1999

Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A (MNK) gene which encodes a copper transporting P-type ATPase (MNK). MNK is normally localized predominantly in the trans-Golgi network (TGN); however, when cells are exposed to excessive copper it is rapidly relocalized to the plasma membrane where it functions in copper efflux. In this study, the c-myc epitope was introduced within the loop connecting the first and second transmembrane regions of MNK. This myc epitope allowed detection of the protein at the surface of living cells and provided the first experimental evidence supporting the common topological model. In cells stably expressing the tagged MNK protein (MNK-tag), extracellular antibodies were internalized to the perinuclear region, indicating that MNK-tag at the TGN constitutively cycles via the plasma membrane in basal copper conditions. Under elevated copper conditions, MNK-tag was recruited to the plasma membrane; however, internalization of MNK-tag was not inhibited and the protein continued to recycle through cytoplasmic membrane compartments. These findings suggest that copper stimulates exocytic movement of MNK to the plasma membrane rather than reducing MNK retrieval and indicate that MNK may remove copper from the cytoplasm by transporting copper into the vesicles through which it cycles. Newly internalized MNK-tag and transferrin were found to co-localize, suggesting that MNK-tag follows a clathrin-coated pit/endosomal pathway into cells. Mutation of the di-leucine, L1487 L1488, prevented uptake of anti-myc antibodies in both basal and elevated copper conditions, thereby identifying this sequence as an endocytic signal for MNK. Analysis of the effects of the di-leucine mutation in elevated copper provided further support for copper+To

stimulated exocytic movement of MNK from the TGN to the plasma membrane. INTRODUCTION The importance of copper as an essential nutrient is evident from the fatal X-linked recessive disorder of copper metabolism, Menkes disease. Affected individuals suffer both severe copper deficiency due to reduced absorption of dietary copper and defective distribution of copper within the body (1). Since copper is an essential component of several enzymes, including dopamine β hydroxylase, lysyl oxidase and cytochrome c oxidase, Menkes patients suffer a range of symptoms including severe neurological problems, connective tissue defects and hypothermia (1). The gene defective in Menkes disease (ATP7A; MNK) encodes a member of a family of proteins, known as P-type ATPases (2–4). These proteins translocate cations through the lipid bilayer of membranes using the energy derived from the hydrolysis of ATP. A role for MNK in copper efflux has come from observations that cultured fibroblasts from Menkes patients accumulate copper (5). More recent evidence is the association of an increased ability to efflux copper with overexpression of the hamster MNK ATPase in Chinese hamster ovary (CHO) cell lines selected for copper resistance (6), and in CHO cell lines stably expressing the human MNK cDNA (7). The MNK protein is localized at the trans-Golgi network (TGN) where it is postulated to deliver copper to copperrequiring enzymes within the secretory pathway, such as lysyl oxidase (8–10). In CHO cells exposed to elevated extracellular copper, MNK rapidly alters its steady-state distribution from the TGN to the plasma membrane, presumably to increase the efficiency of the removal of excess copper from cells (9). The molecular basis for the copper-induced relocalization of MNK to the plasma membrane is unknown. One possibility is that copper stimulates exocytic trafficking of MNK from the TGN to the plasma membrane. An alternative mechanism is that MNK is a constitutively recycling protein and the effect of copper is to inhibit the retrieval of MNK from the plasma

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membrane to the TGN, thereby increasing protein levels at the cell surface. Clearly, an understanding of the trafficking of MNK in basal and copper-stimulated conditions is essential to elucidating the role of the protein in delivering copper to copper-dependent enzymes, and the maintenance of copper homeostasis via copper efflux. This is illustrated in cultured cells from Menkes disease patients where defective localization and copperinduced trafficking of the MNK protein has recently been demonstrated (11). In this study, we introduced an exofacial epitope tag, c-myc, between the first and second membranespanning regions of MNK, to allow detection of surface MNK in living cells. By observing the uptake of anti-myc antibodies in living cells expressing myc-tagged MNK constructs, the internalization of MNK was studied in basal and copperloaded conditions. This experimental system also identified the C-terminal di-leucine, L1487L1488, as an endocytic signal of MNK necesssary for the retrieval of surface MNK into the transferrin-containing endosomal pathway. RESULTS Exofacial tagging of MNK The predicted membrane topology of MNK suggests that the small linker regions between the transmembrane helices are extracellular (12). The longest of these putative extracellular regions is located between the first and second transmembrane domains, and the c-myc epitope (EKQLISEEDL) was introduced into this region immediately after amino acid I693. Immunofluorescence analysis of MNK-tag To establish that the insertion of the myc tag did not alter the distribution of MNK, three independent CHO-K1 cell lines stably expressing the MNK-tag protein were isolated, and its subcellular location was assessed by immunofluorescence using affinity-purified anti-myc antibodies. The results from one cell line, 615D, are shown in Figure 1i. In basal medium, the distribution of MNK-tag was predominantly perinuclear (Fig. 1A), as demonstrated previously for the non-tagged MNK protein (7,9,13). MNK-tag contracted to a tight juxtanuclear location when cells were treated with the drug, brefeldin A (Fig. 1B). This contraction is a well documented effect of brefeldin A on TGN proteins (14,15), and occurs for the non-tagged MNK protein (7,9). The insertion of the myc tag did not affect the ability of copper to stimulate the redistribution of MNK-tag from the TGN, since the exposure of the 615D cells to 200 µM CuCl2 for 2 h induced a shift in MNK-tag distribution from the perinuclear region throughout the cytoplasm to the cell periphery (Fig. 1C). MNK-tag localization was restored to a perinuclear distribution when cells were returned to basal medium for 2 h after a 2 h incubation in elevated copper (Fig. 1D). This indicated that the myc tag did not inhibit the ability of MNK-tag to recycle from the plasma membrane to the TGN. Collectively, these trafficking characteristics of MNK-tag were indistinguishable from those seen previously with the non-tagged normal MNK protein expressed in CHO-K1 cells (7,9,13). The introduction of the myc tag did not appear to impair the copper efflux function since the expression of MNK-tag

Figure 1. The steady-state distribution of MNK and ability to confer copper resistance is not affected by the myc epitope tag. (i) The 615D CHO cells stably expressing the MNK-tag were cultured either in basal medium (A), for 1 h in 10 µg/ml BFA (B), for 2 h in medium containing 200 µM CuCl2 (C) or for 2 h in medium containing 200 µM CuCl2 followed by basal medium for 2 h (D). Cells were fixed and permeabilized and MNK-tag was detected using affinity-purified anti-myc antibodies followed by affinity-purified FITCconjugated sheep anti-mouse IgG. (ii) Colony survival assay showing MNKtag confers resistance to elevated media copper. Untransfected parental CHOK1 cells and 615D cells were exposed to the indicated copper concentrations for 7 days and the surviving colonies scored. The results show the normalized means ± SD of triplicate colony counts for each level of medium copper. Note that at levels of 120 µM CuCl2 and above, the parental CHO-K1 cells failed to form colonies. The copper concentration of basal medium was 0.5 µM.

conferred elevated copper resistance to CHO-K1 cells, as shown in Figure 1ii. A colony survival assay demonstrated the ability of the 615D cell line to survive greater levels of media copper than untransfected parental CHO cells. This resistance profile was similar to that found in previous studies where the ability of the non-tagged MNK protein to confer copper resistance in CHO cells was shown (16). The myc epitope tag is exofacial when MNK-tag is localized at the plasma membrane If the predicted membrane topology of MNK is correct (12), then the myc epitope should be extracellular when MNK-tag is at the plasma membrane. Thus, anti-myc antibodies should readily detect the increased levels of MNK-tag at the surface of non-permeabilized 615D cells grown in elevated copper. To test this hypothesis, 615D cells were cultured on coverslips in

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Figure 2. The myc tag is exofacial when MNK-tag is present at the plasma membrane. Immunofluorescence was used to detect anti-myc antibodies on the surface of 615D cells stably expressing MNK-tag cultured in basal medium (A) or 200 µM CuCl2 (C). This was compared with the localization of the total pool of MNK-tag protein using anti-MNK antibodies following permeabilization in basal medium (B) and 200 µM CuCl2 (D). Anti-myc antibodies bound to the surface of cells were detected using affinity purified anti-mouse FITC-conjugated IgG and the anti-MNK antibodies were visualized using affinity-purified Texas Redconjugated donkey anti-rabbit IgG.

medium containing either basal or elevated copper, fixed with paraformaldehyde without permeabilization of cells, and MNK-tag at the surface was detected with anti-myc antibodies followed by fluorescein isothiocyanate (FITC)–anti-mouse IgG. Following the subsequent permeabilization of cells, antiMNK antibodies followed by Texas Red anti-rabbit IgG were used to detect the total cellular distribution of MNK-tag. The fluorescent signal from anti-myc antibodies bound to the surface of 615D cells grown in basal medium was very low (Fig. 2A). As expected, in the same cells the localization of MNK-tag detected using anti-MNK antibodies was within the perinuclear region (Fig. 2B). In the 615D cells exposed to elevated copper, there was a shift in the steady-state distribution of MNK-tag to the plasma membrane (Fig. 2D). Significantly, this relocalization of MNK-tag to the plasma membrane was coincident with the appearance of a striking fluorescent signal from anti-myc antibodies bound to the surface of these cells (Fig. 2C). This fluorescent signal originated from the myc epitope within MNK-tag, since no signal was detected from anti-myc antibodies with the CHO cells expressing non-tagged MNK protein in basal or elevated copper (data not shown). Thus, the myc epitope located within the loop between the first and second transmembrane domains of MNK is accessible to anti-myc antibodies when the ATPase is present at the plasma membrane of non-permeabilized cells and this represents the first experimental evidence supporting the proposed topology of MNK. MNK-tag constitutively recycles via the plasma membrane in cells in basal medium If MNK-tag constitutively recycles via the cell surface, antimyc antibodies may bind to surface MNK-tag and be internalized from the surrounding medium into intact living cells, as found for other recycling TGN proteins which retrieve

Figure 3. The MNK-tag protein constitutively recycles between the TGN and the plasma membrane in basal medium. Cells stably expressing MNK-tag were incubated in basal medium containing 20 µg/ml of the anti-myc antibody for 1 h (A), 2 h (B), 3 h (C) or 4 h (D) and the internalized anti-myc antibodies were then detected using affinity-purified FITC–anti-mouse IgG after fixing and permeabilizing cells. For the detection of the total pool of MNK-tag protein, the same cells were then incubated with polyclonal rabbit anti-MNK antibodies followed by affinity-purified Texas Red anti-rabbit IgG (E–H). Note that the antimyc antibodies internalized over 3 and 4 h co-localized with the perinuclear pool of MNK-tag protein detected with anti-MNK antibodies.

extracellular antibodies, such as GLUT4, TGN38 and furin (17–19). The 615D cells stably expressing MNK-tag were incubated in basal medium with the anti-myc antibody for various times at 37°C. The cells were then fixed, permeabilized and incubated with FITC anti mouse IgG to visualize any internalized anti-myc antibodies. Timedependent uptake of anti-myc antibodies from basal medium into the 615D cells was observed (Fig. 3). After 1 h, a weak signal from internalized anti-myc antibodies was visible in the perinuclear region of cells (Fig. 3A), and this signal was stronger by 2 h (Fig. 3B). By 3 and 4 h, the perinuclear signal was intense (Fig. 3C and D, respectively). This time-dependent increase in the perinuclear labeling of anti-myc antibodies suggested that the perinuclear pool of MNK-tag protein was constitutively recycling via the cell surface and directing the internalization of anti-myc antibodies from the extracellular medium via the exofacial myc epitope. No signal was detected in untransfected CHO cells nor in cells stably expressing MNK protein without the myc epitope tag (data not shown). The same cells were stained with anti-MNK antibodies followed by Texas Red anti-rabbit IgG to show the perinuclear


Figure 4. MNK-tag is internalized from the plasma membrane in cells exposed to elevated copper. The CHO line, 615D, stably expressing the MNK-tag protein was exposed to 200 µM CuCl2 for 2 h followed by a 4 h incubation in medium containing 200 µM CuCl2 and 20 µg/ml anti-myc antibody. Following fixation and permeabilization of cells, anti-myc antibodies were detected using FITC–anti-mouse IgG (A). To better visualize intracellular compartments labeled by anti-myc antibodies, surface-bound anti-myc antibodies were removed from cells by two washes in acidic buffer prior to fixing and permeabilizing cells (C). In the same cells, the distribution of the total cellular pool of MNK-tag protein was detected using antiMNK antibodies followed by Texas Red-conjugated anti-rabbit IgG (B and D).

distribution of the total cellular pool of MNK-tag protein (Fig. 3E–H). Significantly, after 3 and 4 h the internalized anti-myc antibodies accumulated within perinuclear structures (Fig. 3C and D) that contained the bulk of the total MNK-tag protein pool (Fig. 3G and H). These observations suggested that most, if not all of the MNK-tag protein continually recycles between the TGN and the cell surface in basal medium. A similar timedependent accumulation of anti-myc antibodies was observed for each of the three independent clonal lines expressing MNK-tag (data not shown). MNK-tag constitutively recycles between intracellular compartments and the cell surface when cells are exposed to elevated copper The copper-induced relocalization of MNK to the plasma membrane has been proposed to occur either via increased exocytic trafficking of the protein from the TGN to the cell surface, and/or by inhibition of the endocytic retrieval step of MNK from the plasma membrane to the TGN (7,9,13). To test the latter possibility, we investigated whether anti-myc antibodies were internalized by 615D cells cultured in elevated copper. Cells were pretreated for 2 h with 200 µM CuCl2 to increase levels of MNK-tag at the plasma membrane, anti-myc antibodies were added to the elevated copper medium and then cells were incubated for a further 4 h at 37°C. As seen in Figure 4A, anti-myc antibodies were detected at the plasma membrane and, significantly, there was also strong labeling of intracellular vesicular compartments in the perinuclear region and throughout the cytoplasm. Hence, even though copper stimulated the relocalization of MNK-tag to the plasma membrane (Fig. 4B), the

Figure 5. The L1487L1488-AA mutation in MNK prevents internalization of antimyc antibodies from media containing basal and elevated copper. The CHO line, 736E, stably expressing the MNK(LL-AA)-tag protein was incubated for 4 h in basal medium containing 20 µg/ml anti-myc antibody or for 2 h in medium with 200 µM CuCl2 followed by a 4 h incubation in medium containing both 200 µM CuCl2 and 20 µg/ml anti-myc antibody. The location of anti-myc antibodies was detected using FITC–anti-mouse IgG for cells in basal medium (A) and 200 µM CuCl2 (E). To better visualize any anti-myc antibodies which had internalized, cells were washed with ice-cold acidic buffer to remove surface-bound anti-myc antibodies prior to fixing cells. Following this procedure, FITC–anti-mouse antibodies were used to detect any internal anti-myc antibodies taken up by cells incubated in basal medium (C) or 200 µM CuCl2 (G). In the same cells, the distribution of the total cellular pool of MNK(LL-AA)-tag protein was detected using anti-MNK antibodies followed by Texas Red-conjugated anti-rabbit IgG (B, D, F and H). Note that the perinuclear pool of MNK(LL-AA) protein (B and D) failed to be labeled by surface-presented anti-myc antibodies (A and C).

protein was not maintained at this location but continued to be internalized and recycled through endocytic compartments. After incubating cells in elevated copper medium with anti-myc antibodies, cells were washed in acidic buffer at 4°C to remove surface-bound anti-myc antibodies to allow a clearer demonstration of the internalized anti-myc antibodies. The antimyc antibodies were observed in compartments within both the perinuclear and peripheral regions of the cell (Fig. 4C). This result confirmed that MNK-tag was internalized under conditions of elevated copper and, importantly, demonstrated that inhibition of MNK internalization is not the mechanism by which copper stimulates recruitment of the protein to the plasma membrane. Internalization of MNK requires the L1487L1488 motif We have shown previously that the di-leucine, L1487L1488, is essential for the basal perinuclear localization of the MNK protein,


Figure 6. MNK-tag is internalized to endosomal compartments containing transferrin. The 615D cell line was pre-incubated for 2 h in serum-free medium containing 1% BSA and 200 µM CuCl2 prior to an incubation for the indicated times in serum-free medium containing 1% BSA and 20 µg/ml anti-myc antibody and 100 µg/ml of Texas Red-conjugated human transferrin. Surface-bound anti-myc antibody and transferrin were stripped by two washes in ice-cold acidic buffer and fixed with 4% PFA. Cells were permeabilized and the internalized anti-myc antibodies were detected using affinity-purified FITC–anti-mouse IgG (A, D and G). Internalized transferrin is shown in the same cells (B, E and H), and regions of co-localization with anti-myc antibodies are shown in the merged panels in yellow (C, F and I). Note the extensive co-localization of anti-myc antibodies and transferrin which occurred at uptake times of 5 and 30 min and the reduced colocalization at 60 min.

as mutation of the di-leucine signal results in the accumulation of MNK at the plasma membrane (13). Di-leucines have been shown to direct surface proteins into clathrin-coated pits for endocytosis (20,21). If the L1487L1488 sequence functions in the endocytosis of MNK, then the uptake of anti-myc antibodies should be impaired in cells expressing a myc-tagged form of MNK in which the dileucine is mutated to di-alanine, MNK(LL-AA)-tag. Four CHO cell lines were isolated which stably expressed the MNK(LL-AA)-tag protein. One representative line, 763E, was incubated for 4 h in basal medium containing anti-myc antibodies. The antibodies bound to the periphery of the 763E cells, but significantly, there was a notable absence of intracellular labeling (Fig. 5A). When the surface anti-myc antibodies were removed by acidic washes, no signal was detected when cells were incubated with FITC-antimouse IgG (Fig. 5C). The total pool of MNK(LL-AA)-tag protein was, however, present within the perinuclear region, throughout the cytoplasm and at the plasma membrane (Fig. 5B and D). The failure of surface-presented anti-myc antibodies to be taken up and to label the perinuclear pool of MNK(LL-AA)-tag showed that this internal

pool of protein had not cycled via the cell surface and retrieved extracellular anti-myc antibodies. These results confirm that the dileucine mutation impaired uptake of anti-myc antibodies into those compartments containing the intracellular pool of the MNK(LLAA) protein, and are consistent with the L1487L1488 motif acting as an endocytic targeting signal. Internalization of MNK in elevated copper requires the L1487L1488 signal and suggests that copper induces the exocytic trafficking of MNK To determine whether the di-leucine of MNK was also required for the retrieval of MNK cycling via the plasma membrane under copper-stimulated conditions, the 763E cells were pre-incubated for 2 h in elevated copper, then incubated for 4 h in medium containing both elevated copper and anti-myc antibodies. As found earlier for cells in basal medium (Fig. 5A), anti-myc antibodies appeared to be present only at the plasma membrane (Fig. 5E). This was further supported when no signal was observed after cells were acid-stripped of surface antibody prior to


fixing (Fig. 5G), and suggested that there was no detectable uptake of anti-myc antibodies into intracellular compartments. Thus, the di-leucine, L1487L1488, is necessary for the internalization of MNK in cells in elevated copper conditions, as well as in basal medium. An important difference in the subcellular distribution of the total pool of MNK(LL-AA)-tag was observed in cells cultured in basal medium relative to that in elevated copper. The perinuclear pool of MNK(LL-AA)-tag which was observed in cells in basal medium (Fig. 5B and D), was absent when cells were treated with elevated copper (Fig. 5F and H). This observation suggested that this perinuclear pool of MNK(LL-AA)-tag was stimulated to relocalize when cells were treated with copper. Given that the di-leucine to dialanine mutation impaired internalization from the cell surface, the copper-induced relocalization of MNK(LL-AA)-tag from the TGN suggested that the effect of copper was to increase the exocytic movement of the protein from the TGN, rather than to reduce retrieval from the plasma membrane to the perinuclear compartment. Newly internalized MNK and transferrin co-localize To study the process of internalization of MNK-tag further, we investigated whether internalized anti-myc antibodies were colocalized with newly internalized transferrin, a well-accepted marker for the recycling endocytic pathway. To maximize the levels of MNK-tag leaving the plasma membrane and entering endocytic compartments for trafficking to the TGN, 615D cells growing in elevated copper were returned to basal medium. Anti-myc antibodies and Texas Red-conjugated human transferrin (TRtf) were provided in the basal medium to allow comparison of the distributions of newly internalized anti-myc antibodies and Trtr at various times after uptake. After 5 min both anti-myc antibodies and TRtf were localized within peripheral vesicular structures presumed to be peripheral endosomes (Fig. 6A and B, respectively). Overlaying Figure 6A and B revealed extensive co-localization of internalized MNK-tag with TRtf (Fig. 6C, yellow), suggesting that MNK-tag was internalized and sorted into transferrin-containing endosomes. Within 30 min internalized TRtf was found to be distributed in punctate structures which were concentrated within the juxtanuclear area (Fig. 6E). Transferrin has been shown to migrate from peripheral sorting endosomes to recycling perinuclear endosomes (20,22,23). The internalized MNK-tag was also endocytosed to a perinuclear region within 30 min (Fig. 6D) and showed substantial co-localization with TRtf within this compartment (Fig. 6F, yellow), suggesting that internalized MNK also enters the recycling endosomal compartment. After a 60 min uptake of anti-myc antibodies, most of the MNK-antibody complexes had returned to a more compact perinuclear distribution (Fig. 6G), but their localization only partially overlapped with transferrin (Fig. 6H), with the majority of MNK-tag residing in compartments not containing transferrin (Fig. 6I). Collectively, these data suggested that newly internalized MNK-tag is initially targeted to peripheral and perinuclear endosomes, and is then sorted from these transferrin-containing compartments to the TGN. DISCUSSION The aim of this study was to address the following key questions regarding the cell biology of the MNK copper transporter: (i) does

MNK constitutively cycle between the TGN and the plasma membrane under basal copper conditions?; (ii) is the di-leucine, L1487L1488, an endocytic targeting signal involved in the retrieval of MNK from the plasma membrane to the TGN?; and (iii) does the copper-induced shift in the location of MNK from the TGN to the plasma membrane involve inhibition of MNK endocytosis from the plasma membrane? To answer these questions we introduced the myc epitope tag within the loop connecting the first and second transmembrane domains of MNK. The binding of anti-myc antibodies to this tag in non-permeabilized cells demonstrated that this region of MNK was exofacial and provides the first experimental evidence supporting the proposed topology of MNK. The time-dependent internalization of anti-myc antibodies from the growth medium and accumulation within the perinuclear region of intact cells expressing MNK-tag, suggests that MNK-tag continually cycles between the TGN and the plasma membrane. The perinuclear distribution of accumulated anti-myc antibodies co-localized with most, if not all of the perinuclear pool of MNK-tag. Thus, it is presumably the total pool of MNK-tag that cycles via the plasma membrane, rather than a distinct pool of protein. These data identify MNK as an additional member of the small group of TGN proteins which constitutively cycle between the TGN and the plasma membrane (17–19). In each of the recycling TGN proteins, GLUT4, TGN38 and furin, a tyrosine-based motif or di-leucine signal is necessary for internalization from the plasma membrane (17–19). These types of motif are endocytic targeting signals that direct proteins into clathrin-coated pits prior to endocytosis (20,21). We have recently shown that the L1487L1488 signal of MNK is essential for normal steady-state localization of the protein within the TGN, since MNK lacking an intact di-leucine signal is not localized solely within the perinuclear region, but shifted toward the cytoplasm and the plasma membrane (13) (Fig. 5). The third transmembrane domain of MNK is required for retention of the protein within the TGN (24,25), and this probably accounts for the partial localization of MNK(LLAA)-tag within the perinuclear region. A significant finding in this study was that the perinuclear pool of MNK(LL-AA)-tag was not labeled by anti-myc antibodies supplied to the growth medium of cells. This observation is consistent with the L1487L1488 sequence acting as an endocytic motif which functions in retrieving recycling MNK protein from the plasma membrane to the TGN. This conclusion is further supported by our unpublished observations that a structurally different internalization signal from the TGN38 protein, YQRL, can function place of the L1487L1488 sequence to retrieve MNKtag from the plasma membrane. During the preparation of this manuscript the ability of the MNK C-terminal region to internalize a reporter protein from the plasma membrane was demonstrated and this was dependent on the L1487L1488 dileucine signal (26). These findings, together with our antibody uptake experiments with the full length MNK protein, suggest that L1487L1488 is both sufficient and essential for retrieval of MNK from the plasma membrane. MNK-tag and transferrin were co-localized within peripheral compartments at early uptake times, and within the juxtanuclear region after longer periods, suggesting that after internalization of MNK via L1487L1488, the protein follows the transferrincontaining recycling endosomal pathway (Fig. 6). But at 60 min uptake, the predominant perinuclear distribution of internalized


anti-myc antibodies did not co-localize with the steady-state transferrin distribution as extensively as at the earlier times. Thus at later time points there was the eventual sorting of MNK-tag from the transferrin-containing endosomal compartments to the TGN. It is not known whether the L1487L1488 signal influences these later sorting processes, but recent studies have shown that the transmembrane domain of TGN38, which is involved in TGN retention, also plays a role in sorting recycling protein from endosomal compartments to the TGN (27). Therefore, in addition to L1487L1488, there is the potential for other regions of MNK to influence the later sorting steps from endosomes to the TGN. The ability to exofacially label MNK and track its endocytic movement to the TGN provides the necessary tools for identifying these sequences in future experiments. A key aspect to the function of the MNK protein in regulating copper levels in the cell is its relocalization from the TGN to the plasma membrane when cells are exposed to elevated copper (7,9,13). The mechanism of this process is not understood, but two hypotheses have been proposed (9). The first is that copper increases the exocytic rate of MNK trafficking from the TGN to the plasma membrane; the second proposes that the internalization of MNK recycling from the plasma membrane is reduced by copper. Our results support the first hypothesis. When anti-myc antibodies were supplied to cells grown in elevated copper, the antibodies were internalized (Fig. 4), showing that MNK-tag internalization is not inhibited by elevated copper, although a slight reduction in rates of endocytosis cannot be excluded. Therefore, even under elevated copper conditions, when MNK is functioning to maintain copper homeostasis, it is not a static surface protein involved in effluxing copper from this location, but is constantly recycling. Given this situation, it is reasonable to suggest that copper efflux by MNK may occur both via the pool of surface protein, and by pumping copper into the cytoplasmic vesicular compartments as it recycles; these vesicles then expel their copper load upon fusion with the plasma membrane. Such a proposal for copper efflux is reminiscent of that proposed for the mechanism of copper transport by the Wilson protein in hepatocytes (28). An important finding in this study is that elevated copper resulted in the relocalization of the perinuclear pool of MNK(LLAA)-tag protein present in basal medium (Fig. 5B and F). Since the di-leucine mutation completely impairs internalization of antimyc antibodies in both basal and elevated copper (Fig. 5C and G), the copper-induced relocalization of the perinuclear pool of MNK(LL-AA)-tag could not be the result of reduced internalization of surface MNK(LL-AA)-tag to the perinuclear region. Rather, the data provide further evidence that the copperinduced relocalization of MNK from the TGN to the plasma membrane involves increased exocytosis of the protein from the TGN. In summary, the exofacial tagging of the Menkes ATPase has proven to be a powerful tool in the analysis of the trafficking of the protein in basal and copper-induced states. The ability to label surface MNK protein has demonstrated that the di-leucine, L1487L1488, is essential for internalization of MNK from the plasma membrane into the endosomal pathway. This experimental system has also provided the first evidence for copper-stimulated exocytic movement of MNK from the TGN. Whereas the biological basis for recycling of MNK in elevated copper is presumably to remove copper from the cell, the basis for the recycling of MNK in basal medium remains to be shown.

Future studies will need to determine whether this process is simply a corrective mechanism to retrieve protein which has escaped retention in the TGN, or whether recycling in basal medium represents low level copper-induced trafficking of MNK by basal copper. It is also possible that basal recycling of MNK represents the proposed role of the protein in delivering copper to cuproenzymes in the secretory pathway, which may occur within a class of vesicles which are functionally and physically distinct from those through which MNK cycles under copper-induced conditions. Determining whether mutations that we have recently identified which inhibit the copper-induced relocalization of MNK toward the plasma membrane (11,16,29) also impair constitutive recycling via the cell surface, will provide avenues for testing these possibilities. MATERIALS AND METHODS Construction of a myc-tagged MNK cDNA PCR was used to introduce the c-myc epitope, EKQLISEEDL, immediately after amino acid I693. A reverse primer containing the coding sequence for the myc epitope was used in PCR on a plasmid template containing the MNK cDNA, pCMB19 (30). A 300 bp SacII/HaeII PCR product with the myc-tag coding sequence was introduced into the MNK cDNA replacing the corresponding region of the normal MNK cDNA using standard molecular biology techniques (31). DNA sequencing using the Thermo Sequenase kit (Amersham Pharmacia Biotech, Cleveland, OH) was used to ensure that erroneous mutations had not been introduced during PCR and to confirm the in-frame insertion of the myc tag coding sequence into the MNK cDNA. The myc-tagged MNK cDNA was inserted into the mammalian expression vector, pCMB77 (13), to generate the plasmid pMNKtag. A myc-tagged MNK cDNA construct, in which L1487L1488 was replaced by AA to generate pMNK(LL-AA)-tag, was created by replacement of the MNK C-terminal coding region of pMNKtag with the corresponding region of pMNK(L1487L1488-AA) described previously (13). Cell culture and transfection experiments Plasmid DNA was prepared using Qiagen (Clifton Hill, Australia) midi columns and CHO-K1 cells were stably transfected using Lipofectamine (Gibco, Gaithersburg, MD). G418-resistant cell colonies were screened for MNK-tag or MNK(LL-AA)-tag expression by indirect immunofluorescence in 96 well trays. This resulted in the isolation of three cell lines stably expressing the MNK-tag protein, and four cell lines stably expressing MNK(LLAA)-tag. Cells were maintained at 37°C in Eagle’s basal medium supplemented with 10% fetal calf serum (Trace Biosciences, Castle Hill, Australia), 20 mM L-proline and 0.2% (w/v) bicarbonate. Basal medium had a concentration of 0.5 µM Cu and where indicated, 200 µM CuCl2 was added to medium. Brefeldin A (Sigma, St Louis, MO) (10 µg/ml) was added to the growth medium for 1 h. Transient transfections of CHO-K1 cells were performed as previously described (13) using Lipofectamine (Gibco). Colony survival assay The colony survival assay was essentially the same as that used previously to determine survival of cells in elevated copper


(16). One hundred cells per petri dish were seeded in triplicate and allowed to attach overnight prior to a 7 day incubation in growth medium containing 0.5–200 µM CuCl2. Cell colonies were fixed in 90% methanol and 10% formaldehyde, stained with Giemsa stain (Merck, Kilsyth, Australia) and counted. Indirect immunofluorescence Immunofluorescence analysis of cells was carried out essentially as described previously (9,13). Cells were seeded onto 13 mm round glass coverslips 48 h prior to immunofluorescence. Coverslips were washed with ice-cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) for 20 min. Coverslips were then permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed several times with PBS and then blocked overnight in PBS containing 1% bovine serum albumin (BSA)/1% gelatin. Where indicated, MNK-tag and MNK(LL-AA)-tag proteins were detected by incubating coverslips with a sodium sulphate preparation of antiMNK polyclonal rabbit antibodies diluted 1:500 in 1% BSA/1% gelatin. These antibodies were raised to the MNK N-terminal region and have been described previously (9). Anti-MNK antibodies were visualized using Texas Red-conjugated donkey anti-rabbit IgG antibodies (Jackson Laboratory, Bar Harbor, ME) diluted 1:100 in 1% BSA/1% gelatin. Where indicated, MNK-tag protein was also detected in cells using 20 µg/ml of the affinity-purified monoclonal anti-myc antibody (9E10; Boehringer Mannheim, Mannheim, Germany) and visualized using affinity-purified FITC-conjugated sheep anti-mouse IgG antibodies (Silenus) diluted 1:200 in 1% BSA/1% gelatin. Coverslips were mounted using 2.6% DABCO [1,4-diazabicyclo-(2.2.2) octane] (Sigma) in 90% glycerol. Coverslips were analysed using a Zeiss Axioskop microscope using a 100× oil objective and digitized images were captured using a Photometrics imagepoint CCD camera. Photographic exposure times and printing conditions were controlled for all images. Antimouse and anti-rabbit secondary antibodies did not show detectable cross-reactivity to anti-MNK (rabbit) and anti-myc (mouse) antibodies, respectively (data not shown). Antibody uptake detected by immunofluorescence Cells grown on glass coverslips were incubated at 37°C for indicated times in growth medium containing 20 µg/ml of the affinity-purified anti-myc monoclonal antibody, washed several times with ice-cold PBS, then fixed using 4% PFA for 20 min. Where indicated, anti-myc antibodies were stripped from the surface of intact living cells prior to fixing, using a previously described procedure (17) whereby coverslips were washed twice for 2 min in ice-cold acidic buffer (100 mM glycine, 20 mM magnesium acetate, 50 mM potassium chloride, pH 2.2). Cells were fixed and then permeabilized with 0.1% Triton X-100 for 10 min. Anti-myc antibodies were detected using affinity-purified FITC-conjugated sheep antimouse IgG. For the detection of the total cellular pool of myctagged MNK, coverslips were incubated with anti-MNK polyclonal rabbit antibodies, and detected using Texas Redconjugated donkey anti-rabbit IgG. Coverslips were mounted and analysed as described above. Uptake of anti-myc antibodies and transferrin CHO-K1 cells stably overexpressing the MNK-tag protein were cultured on glass coverslips and then incubated in

elevated copper for 2 h to enrich the levels of MNK-tag at the plasma membrane. Cells were transferred to 37°C serum-free medium containing 1% BSA without elevated copper containing 20 µg/ml anti-myc antibodies and 100 µg/ml Texas Red-conjugated human transferrin (Trtf) (Molecular Probes, Eugene, OR) for the indicated times to allow uptake of the antibodies and transferrin. Cells were washed twice for 2 min in ice-cold acidic buffer (as described above) to remove both the anti-myc antibodies and transferrin bound to the cell surface, and then washed twice with ice-cold PBS. Cells were fixed and permeabilized, as described above, and anti-myc antibodies were visualized by incubating cells with FITCconjugated sheep anti-mouse IgG (1:200). Coverslips were mounted and analyzed as described above. ACKNOWLEDGEMENTS We would like to thank Sharon La Fontaine, Daniel Strausak, Loreta Ambrosini and James Camakaris for helpful scientific discussions and for reading the manuscript. This work was supported by funding from the National Health and Medical Research Council and the International Copper Association. REFERENCES 1. Danks, D.M. (1995) Disorders of copper transport. In Scriver, C.R., Beaudet, A.L., Sly, W.M. and Valle, D. (eds), The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill, New York, NY, pp. 2211–2235. 2. Chelly, J., Turmer, Z., Tonneson, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N. and Monaco, A.P. (1993) Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nature Genet., 3, 14–19. 3. Mercer, J.F.B., Livingston, J., Hall, B., Paynter, J.A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak, D. and Glover, T.W. (1993) Isolation of a partial candidate gene for Menkes disease by positional cloning. Nature Genet., 3, 20–25. 4. Vulpe, C., Levinson, B., Whitney, S., Packman, S. and Gitschier, J. (1993) Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nature Genet., 3, 7–13. 5. Herd, S.M., Camakaris, J., Christofferson, R., Wookey, P. and Danks, D.M. (1987) Uptake and efflux of copper-64 in Menkes’-disease and normal continuous lymphoid cell lines. Biochem. J., 247, 341–347. 6. Camakaris, J., Petris, M.J., Bailey, L., Shen, P., Lockhart, P., Glover, T.W., Barcroft, C.L., Patton, J. and Mercer, J.F.B. (1995) Gene amplification of the Menkes (MNK; ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux. Hum. Mol. Genet., 4, 2117–2123. 7. La Fontaine, S., Firth, S.D., Lockhart, P.J., Brooks, H., Parton, R.G., Camakaris, J. and Mercer, J.F.B. (1998) Functional analysis and intracellular localization of the human Menkes protein (MNK) stably expressed from a cDNA construct in Chinese Hamster Ovary cells (CHOK1) Hum. Mol. Genet., 7, 1293–1300. 8. Dierick, H.A., Adam, A.N., Escara-Wilke, J.F. and Glover, T.W. (1997) Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network. Hum. Mol. Genet., 6, 409–416. 9. Petris, M.J., Mercer, J.F.B., Culvenor, J.G., Lockhart, P., Gleeson, P.A. and Camakaris, J. (1996) Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. EMBO J., 15, 6084–6095. 10. Yamaguchi, Y., Heiny, M.E., Suzuki, M. and Gitlin, J.D. (1996) Biochemical characterization and intracellular localization of the Menkes disease protein. Proc. Natl. Acad. Sci. USA, 93, 14030–14035. 11. Ambrosini, L. and Mercer, J.F.B. (1999) Defective copper-induced trafficking and localization of the Menkes protein in patients with mild and copper-treated classical Menkes disease. Hum. Mol. Genet., 8, 1547– 1555.


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