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Human Molecular Genetics Pages 2063-2071  

A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network
Introduction
Results
Discussion
Materials And Methods
   Affinity-purified MNK antibody
   Site directed in vitro mutagenesis and transient expression of MNK constructs
   Isolation of stably expressing MNK cells lines
   Indirect immunofluorescence microscopy
   Preparation of plasma membrane lawns
   Western blots
   Copper accumulation measurements
Acknowledgements
Abbreviations
References

A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network

A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network

Michael J. Petris1,2, James Camakaris2,+, Mark Greenough2, Sharon LaFontaine1 and Julian F. B. Mercer3,+,*

1The Murdoch Institute, Royal Children's Hospital and 2Department of Genetics, University of Melbourne, Parkville 3052, Australia and 3Centre for Cellular and Molecular Biology, Deakin University, Clayton 3168, Australia

Received July 7, 1998; Revised and Accepted September 10, 1998

The human X-linked recessive disorder of copper metabolism, Menkes disease, is caused by a defect in the MNK (ATP7A) gene which encodes a transmembrane copper-transporting P-type ATPase (MNK). MNK is an important component of the mammalian copper transport pathway, and previous studies in cultured cells have localized MNK to the final compartment of the Golgi apparatus, the trans-Golgi network (TGN). At this location, MNK is predicted to supply copper to copper-dependent enzymes as they migrate through the secretory pathway. However, under conditions of elevated extracellular copper, the MNK protein undergoes a rapid relocalization to the plasma membrane where it functions in the efflux of copper from cells. In this study, three di-leucine motifs and a cluster of four acidic amino acids within the C-terminal region of MNK were investigated as candidate signals necessary for steady-state TGN localization. In vitro mutagenesis of the human MNK cDNA and immunofluorescence detection of mutant forms of MNK expressed in cultured cells demonstrated that the di-leucine, L1487L1488, was essential for localization of MNK within the TGN, but not for copper efflux. We suggest that this di-leucine motif is a putative endocytic targeting motif necessary for the retrieval of MNK from the plasma membrane to the TGN. Our data, along with the recent demonstration that the third transmembrane region of MNK functions as a TGN targeting signal, suggests that MNK localization to the TGN may be a two-step process involving TGN retention via the transmembrane region, and recycling to this compartment from the plasma membrane via the L1487L1488 motif.

INTRODUCTION

Menkes disease is an X-linked recessive disorder of copper metabolism which is lethal during early childhood. Affected males suffer a systemic copper deficiency due to malabsorption and defective distribution of dietary copper, and the ensuing clinical presentations include severe neurological problems, connective tissue defects and hypothermia (1). These pleiotropic symptoms of Menkes disease can be ascribed to the reduced activities of a range of copper-dependent enzymes, which include cytochrome c oxidase, dopamine [beta]-hydroxylase and lysyl oxidase (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 membranes using the energy derived from the hydrolysis of ATP. The features of the predicted MNK protein suggest that it may be involved in copper efflux from cells, which is consistent with the observations that cultured fibroblasts from Menkes patients accumulate copper (5). Further evidence to support a role for MNK in copper efflux has come from our studies of copper-resistant Chinese hamster ovary (CHO) cell lines, which have an increased ability to efflux copper due to elevated expression of the hamster MNK homologue (6). Immunofluorescence analysis of these cell lines showed that the hamster MNK protein has a steady-state localization at the trans-Golgi network (TGN) of cells (7). The localization of MNK to this compartment of the Golgi apparatus has also been reported in normal CHO cells (8) and in human cultured cells (9). It is thought that MNK may function to transport copper across the membranes of the TGN in cells grown under basal copper conditions and supply copper to copper-dependent enzymes as they migrate through the biosynthetic pathway (7-9). The reduced activities of the copper-dependent enzyme, lysyl oxidase, in cultured Menkes fibroblasts is consistent with this suggested role of MNK (10). The MNK protein in the CHO variants rapidly alters its steady-state localization from the TGN towards the plasma membrane when cells are exposed to elevated copper in the culture medium, and recycles back to the TGN from the cell surface when basal copper levels are restored (7). We recently have demonstrated that the TGN localization and copper-induced redistribution to the plasma membrane occurs for the human MNK protein expressed in CHO cells (11). The recycling ability of MNK appears to occur constitutively in cells cultured in medium without added copper (7).

Identification of the molecular signals within the MNK protein which target the protein to the TGN, sort the protein into vesicles for trafficking to the plasma membrane and internalize surface-located protein for retrieval to the TGN will be important in elucidating the function of the protein, and may provide insights into the molecular basis of the Menkes disease phenotype in those patients in whom the protein is expressed. Signals which confer the intracellular localization of membrane proteins have been identified in extracellular domains, transmembrane domains and in cytoplasmic regions. The transmembrane domain of different glycosyltransferases is involved in their retention within the Golgi (12,13). Recently, the third transmembrane region has been shown to be essential for TGN localization of MNK (14,15), and this domain is sufficient to confer TGN localization to a reporter protein not normally located in this compartment (15). Given that MNK constitutively recycles between the TGN and the cell surface (7), we hypothesize that TGN localization of MNK will also depend on retrieval of surface MNK protein into the endocytic pathway for recycling to the TGN. Endocytic signals are generally cytoplasmic and consist of short amino acid sequences which commonly fall into two classes, tyrosine-based motifs and di-leucine (or leucine-isoleucine) signals (16,17). Tyrosine-based motifs contain a critical tyrosine residue within the consensus sequences, NPXY or YXXØ (where Ø represents an amino acid with a bulky hydrophobic group). Both tyrosine-based motifs and di-leucine signals function by concentrating proteins into clathrin-coated pits at the cell surface prior to endocytosis. Tyrosine- or di-leucine-based targeting motifs are essential for conferring the steady-state TGN localization by surface retrieval of a small number of recycling proteins including TGN38 (18), the endopeptidase, furin (19, 20), the envelope protein of varicella-zoster virus, gp1 (21), and GLUT4 (22, 23). It has also been shown that clusters of acidic amino acids located downstream of the tyrosine-based motif of furin and gp1 are involved in conferring localization to the TGN for these recycling proteins (19-21).

Based on our hypothesis that TGN localization of MNK relies upon a mechanism of retrieval from the cell surface, we searched the predicted cytoplasmic regions of MNK for sequences which resembled the classes of endocytic signals described above. This search led to the identification of three di-leucines and a sequence of four acid amino acids within the C-terminal 50 amino acids of the MNK protein (Fig. 1), and these sequences are also conserved in the MNK homologues of rodents (24,25). The mutation of a retrieval signal within MNK would be predicted to result in increased levels of MNK at the plasma membrane, as occurs when the internalization signal of the above TGN proteins is mutated (18-23). In this report, in vitro mutagenesis was used to demonstrate that the di-leucine sequence, L1487L1488, was essential for the steady-state localization of MNK within the TGN, but not for copper efflux function.


Figure 1. MNK C-terminal truncations and amino acid substitution mutations used in this study. The amino acid sequence of the C-terminal region of MNK is shown, with candidate TGN targeting motifs underlined (see text). Amino acids substituted for alanine residues are shown in bold type.

RESULTS

To investigate the significance of the C-terminal sequences in maintaining steady-state TGN localization of MNK, we used a plasmid construct containing the 4.6 kb coding sequence of the MNK cDNA, which had been shown to express the MNK protein stably in CHO-K1 cells (11). To investigate a potential targeting role for the three di-leucines, stop codons were introduced to truncate MNK before each of these motifs (Fig. 1). The effect of these MNK deletions on localization was investigated by transient expression of the mutated MNK proteins in CHO-K1 cells and detected by indirect immunofluorescence. Transient expression of the wild-type MNK protein, MNK(wt), showed that the protein was located predominantly to the perinuclear region in basal medium, with no apparent staining of the cell periphery (Fig. 2A). The addition of 12 µg/ml (189 µM) copper to the growth medium induced an altered localization of MNK so that it was now present within the perinuclear region, throughout the cytoplasm and the cell periphery (Fig. 2B). These observations were entirely consistent with the basal TGN localization of MNK and the copper-induced increases in MNK levels at the plasma membrane reported for the stably expressed MNK protein in CHO cells (11) and the endogenous MNK protein in copper-resistant CHO cells (7). In control experiments, transfection of the expression vector alone resulted in no change to the low level background fluorescence observed in non-transfected CHO cells (data not shown). Thus, the transient expression system was a suitable system for investigating the localization of the truncated forms of the MNK protein.


Figure 2. Immunofluorescence microscopy of MNK C-terminal truncations transiently expressed in CHO-K1 cells. CHO-K1 cells transfected with the indicated construct were cultured in basal medium (1 µM Cu) for 24 h (A, C, E and G) or basal medium for 23 h followed by 1 h in 189 µM Cu (B, D, F and H), and were fixed in acetone. Immunofluorescence detection was performed by incubation with affinity-purified MNK antibodies and affinity-purified FITC-conjugated sheep anti-rabbit IgG antibodies. As expected in cells in basal medium, the MNK(wt) protein was concentrated within the TGN (perinuclear region) and there was no labelling detected at the plasma membrane (A), whilst in elevated copper the MNK(wt) protein was perinuclear, throughout the cytoplasm and at the plasma membrane (B). Note that each of the truncated forms of MNK in basal medium was present within the perinuclear region, throughout the cytoplasm and at the cell periphery (C, E and G), and this distribution clearly differed from the predominantly perinuclear labelling of the MNK(wt) (A). The localization of the MNK truncations in copper-treated cells (D, F and H) was indistinguishable from that in untreated cells.

The distributions of MNK([Delta]1452-1500), MNK([Delta]1466-1500) and MNK([Delta]1486-1500) (Fig. 2C, E and G, respectively) differed markedly from that of the MNK(wt) protein (Fig. 2A) in cells grown in basal medium. For each of these truncated forms of MNK, immunofluorescent staining was observed within the perinuclear region and throughout the cytoplasm and, significantly, there was strong labelling of the cell periphery. Thus, with MNK([Delta]1452-1500), MNK([Delta]1466-1500) and MNK([Delta]1486- 1500) proteins there was a clear shift in steady-state localization from the TGN towards the cell periphery. The distribution of MNK([Delta]1452-1500), MNK([Delta]1466-1500) and MNK([Delta]1486- 1500) proteins in CHO-K1 cells grown in elevated copper concentrations (Fig. 2D, F and H, respectively) was indistinguishable from the distribution observed in basal medium described above and from the copper relocalization effect on the wild-type MNK protein (Fig. 2B). From these results, it was concluded that the region downstream of H1485 was necessary for the localization of the MNK protein in basal medium to a predominantly perinuclear location.

The nonsense mutations described above demarcate the sequence 1485HSLLVGDFREDDDTAL1500 as essential for steady-state localization of MNK at the TGN. This sequence contains two signals which are similar to those endocytic motifs identified in other proteins as essential for TGN localization: the di-leucine, L1487L1488, and the downstream acidic sequence, 1494EDDD1497. To test the possible influence of regions downstream of L1487L1488, this region was deleted from MNK by introducing a nonsense mutation to truncate MNK immediately after the L1487L1488 to create the protein MNK([Delta]1489-1500) (Fig. 1). A second mutation to test directly the importance of the acidic sequence was also generated by substitution of alanine for glutamate and aspartate residues to yield MNK(1494AAAA1497). The subcellular localization of MNK([Delta]1489-1500) (Fig. 3C) and MNK(1494AAAA1497) (Fig. 3E) in cells grown in basal medium was similar to that of the MNK(wt) protein (Fig. 3A), as evidenced by a predominant perinuclear distribution with no staining of the cell periphery. The copper-induced relocalization of MNK towards the plasma membrane was observed for both MNK([Delta]1489-1500) (Fig. 3D) and MNK(1494AAAA1497) (Fig. 3F), as demonstrated by the strong labelling at the cell periphery, and was similar to the effect of copper on the MNK(wt) protein (Fig. 3B). Hence, these data suggest that the region downstream of L1488 is not required for the normal steady-state TGN localization of MNK, nor for the trafficking response of the protein to elevated copper.


Figure 3. Immunofluorescence microscopy of CHO-K1 cells transiently expressing MNK C-terminal mutations. CHO-K1 cells were transfected with the indicated MNK construct and fixed in acetone after culturing for 24 h in basal medium (A, C, E, G, I and K) or 23 h in basal medium followed by 1 h in medium containing 189 µM copper (B, D, F, H, J and L). MNK protein was detected by incubating acetone-fixed cells with affinity-purified MNK antibodies followed by affinity-purified FITC-conjugated sheep anti-rabbit IgG antibodies. The MNK([Delta]1489-1500) and MNK(1494AAAA1497) mutations had no apparent effect on localization, as determined by the perinuclear distribution in basal medium (C and E) and the marked increase in labelling of the cell periphery in copper-treated cells (D and F). Note that the basal localization of MNK(L1487-A), MNK(L1488-A) and MNK(L1487L1488-AA) was not predominantly perinuclear as for MNK(wt) (A), and for each mutant there was labelling within the perinuclear region, throughout the cytoplasm and at the cell periphery (G, I and K). The distribution of all forms of MNK was essentially equivalent in copper-treated cells.

The observation that in basal medium the MNK([Delta]1486-1500) protein was shifted towards the plasma membrane and the MNK([Delta]1489-1500) protein was TGN-localized defined the sequence 1486SLL1488 as essential for the steady-state TGN localization of MNK. The importance of the di-leucine motif in this sequence in conferring TGN localization of MNK was tested by substituting alanine for the leucines. When L1487 and L1488 were mutated individually to alanine to yield MNK(L1487-A) and MNK(L1488-A), the resulting distribution in basal medium (Fig. 3G and I, respectively) was shifted markedly towards the cell periphery relative to that of the MNK(wt) protein which showed no plasma membrane labelling (Fig. 3A). No apparent difference of this distribution in basal medium occurred when cells expressing MNK(L1487-A) or MNK(L1488-A) were incubated in elevated copper (Fig. 3H and J, respectively). When both leucines were mutated to alanine, as in MNK(L1487L1488-AA), strong labelling of the cell periphery was observed in basal medium (Fig. 3K). As seen previously for the other forms of MNK with altered localization, the distribution of MNK(L1487L1488-AA) in cells in elevated copper appeared indistinguishable from that in cells in basal medium (Fig. 3L).

The results with the MNK(L1487L1488-AA) protein suggested an increased association of this protein with the plasma membrane compared with MNK(wt) in cells cultured in basal medium. These observations were confirmed by immunofluorescence labelling of MNK associated with isolated fragments of the plasma membrane (PM lawns) of cells transiently expressing either MNK(wt) or MNK(L1487L1488-AA). The technique for isolating PM lawns from cells involved sonication of cells cultured on glass coverslips to yield plasma membrane fragments with their cytoplasmic surface exposed and which are free of intracellular membranes (26,27). We have used this technique previously to demonstrate increased levels of the hamster MNK protein associated with the plasma membrane when copper-resistant CHO cells were exposed to elevated copper (7). Using the MNK antibodies, labelling of PM lawns from cells expressing the MNK(wt) protein was not detected when cells were cultured in basal medium (Fig. 4A), whilst, as expected, there was strong labelling of plasma membrane fragments when cells were exposed to elevated copper (Fig. 4B). Significantly, there was strong labelling of the MNK(L1487L1488-AA) protein in PM lawns derived from cells cultured in basal medium (Fig. 4C). This observation suggested that the effect of the L1487L1488-AA mutation was to shift the localization of MNK towards the plasma membrane.


Figure 4. The MNK(L1487L1488-AA) mutation results in re-localization of MNK towards the plasma membrane. PM lawns were isolated from CHO-K1 cells transiently expressing either the MNK(wt) (A and B) or MNK(L1487L1488-AA) (C) proteins in either basal medium (A and C) or 189 µM copper (B). The MNK protein was then detected using affinity-purified MNK antibodies and affinity-purified FITC-conjugated sheep anti-rabbit IgG antibodies. Note that the strong labelling of PM lawns occurred for MNK(L1487L1488-AA) in cells cultured in basal medium (C), but not for MNK(wt) (A), which was detected in PM lawns only after cells were exposed to elevated copper (B).

It previously has been documented that overexpression of membrane proteins in transfected cells can result in the saturation of sorting and localization mechanisms and result in diversion of the protein to the cell surface (28,29). In our experiments where mutated forms of MNK were transiently expressed, it could be argued that for those mutants which showed cell surface labelling there was an elevated level of protein expression, to extents where mechanisms for TGN targeting were saturated. This is unlikely given that there was a broad range of expression levels obtained in the population of transiently transfected cells, and that for each mutant form of MNK, the distribution of the protein was similar across the population. Furthermore, quantitation of immunofluorescence levels from transiently transfected cells using confocal microscopy has indicated that the altered localization of the mutant forms of MNK was not due to elevated expression relative to MNK(wt) (data not shown). However, to investigate more rigorously the possibility that altered localization of MNK was caused by overexpression, we isolated four independent CHO cell lines stably expressing the MNK(L1487L1488-AA) protein. Within each of these cell lines, the subcellular distribution of the MNK(L1487L1488-AA) protein was the same as that seen in the transiently transfected cells. One representative clone is shown in Figure 5A, with strong labelling throughout the cytoplasm and cell periphery. A CHO cell line stably expressing the MNK(wt) protein was also isolated and showed the characteristic perinuclear TGN localization of MNK in basal medium (Fig. 5A), as we have demonstrated previously in several clonal lines with a range of expression levels (11). The MNK expression levels within the MNK(wt) and MNK(L1487L1488-AA) clones shown in Figure 5A were approximately equivalent, as indicated by the western blot analysis (Fig. 5B). Other MNK(wt) clones with expression levels substantially elevated above those shown for the MNK(L1487L1488-AA) clone shown in Figure 5 have been isolated, and maintain predominant TGN localization (data not shown). Overall, the above observations negate overexpression of the mutant forms of MNK as the cause of the altered localization towards the plasma membrane, and suggest that the L1487L1488 signal is essential in conferring localization of MNK to the TGN.


Figure 5. (A) Immunofluorescence detection of MNK(wt) and MNK(L1487L1488-AA) stably expressed in CHO-K1 cells. Cells were cultured in basal medium for 48 h, fixed in acetone and processed for immunofluorescence using affinity-purified MNK antibodies and affinity-purified FITC-conjugated sheep anti-rabbit IgG antibodies. (B) Western blot analysis comparing MNK expression levels in non-transfected parental CHO-K1 cells, and the stably expressing clones of MNK(wt) and MNK(L1487L1488-AA) shown in (A). Each lane contained 75 µg of protein and was detected using affinity-purified MNK antibodies and enhanced chemiluminescence.

To determine whether the L1487L1488-AA mutation affected the copper efflux function of the MNK protein, we compared the levels of copper which accumulated within the stable lines of MNK(L1487L1488-AA), MNK(wt) and non-transfected parental CHO-K1 cells. When these cells were exposed for 20 h to three levels of extracellular copper, the MNK(wt) clone accumulated less copper than the parental CHO-K1 cell line (Fig. 6), as expected from previous studies (6,11). The MNK(L1487L1488-AA) clone also showed reduced intracellular copper relative to parental CHO-K1 cells over each of the three extracellular copper concentrations. These data suggest the L1487L1488-AA mutation did not compromise the ability of the MNK(L1487L1488-AA) protein to efflux copper. Indeed, the MNK(L1487L1488-AA) clone consistently maintained lower intracellular copper levels than the MNK(wt) clone over the range of media copper concentrations (Fig. 6), at comparable levels of MNK expression present within both cell lines (Fig. 5B).


Figure 6. Analysis of the accumulation of 64Cu in cells stably expressing MNK(wt) or MNK(L1487L1488-AA) protein relative to non-transfected cells. Cells were cultured for 20 h in media containing the indicated concentration of cold copper and carrier-free 64Cu. The bars represent the percentages of intracellular copper relative to that within the parental CHO-K1 cell line at20 h in 250 µM extracellular Cu. Percentages were averaged from duplicate measurements, the values of which differed by no more than 15%.

DISCUSSION

It has been demonstrated previously that the MNK protein is localized predominantly to the TGN in basal media, and may constitutively recycle between this compartment and the plasma membrane (7-9). In glycosyltransferases, Golgi localization is conferred by active retention via the transmembrane domain (12,13). The recent demonstration that the third transmembrane region of MNK confers TGN localization to a reporter protein suggests that this region contains information for retaining MNK within the TGN as it migrates through the biosynthetic pathway (15). Given that MNK is a recycling protein, we proposed that the maintenance of a TGN localization for MNK involves a second mechanism whereby MNK protein which has migrated to the cell surface is constitutively retrieved to the endocytic pathway for return to the TGN. Tyrosine-based motifs or di-leucine sequences often are involved in the retrieval of surface proteins into clathrin-coated vesicles for the subsequent delivery to endosomal compartments. The process of constitutive retrieval from the plasma membrane via these motifs is involved in maintaining the TGN localization of TGN38, furin, gp-1 and GLUT-4 (18-23). By analogy, it was postulated that the exclusion of MNK from the cell surface and the maintenance of steady-state TGN localization may be conferred by one or more of these types of targeting signal. An analysis of the MNK protein sequence led to the identification of three di-leucines and an acidic sequence, within the last 50 amino acids, as candidates for mediating TGN localization.

The amino acids L1487 and L1488 were identified as essential in conferring steady-state TGN localization for the MNK protein since, when these amino acids were deleted or mutated to alanine, there was a substantial increase in the level of MNK protein at the plasma membrane. Given that it is well documented that di-leucine signals are involved in retrieval of surface proteins into the endocytic pathway (22,23,29-32), we propose that L1487 and L1488 form an indirect TGN targeting signal which acts to internalize MNK at the plasma membrane and direct the protein to the TGN along the endocytic arm of the recycling pathway. It was notable that mutation of the L1487L1488 signal did not fully abolish perinuclear labelling of MNK. This was probably due to retention of newly synthesized MNK in the TGN via the third transmembrane domain, recently identified as containing a putative TGN retention signal (15). Collectively, the current data suggest that the TGN localization of MNK occurs via two steps, the first involving direct retention in this compartment via the transmembrane region, and the second occurring by retrieval of recycling MNK from the plasma membrane via the di-leucine signal. This model is similar to that for the TGN38 protein, which has a transmembrane signal essential for TGN localization (33) and a tyrosine-based endocytic signal for the retrieval of surface protein (18).

The di-leucine signal, L1487L1488 of MNK is present at an equivalent position within the C-terminus of the MNK homologues of mouse (24), rat (25) and the Tammar wallaby, an Australian marsupial (M. Howie, personal communication). Notably, the MNK homologue of Caenorhabditis elegans (nematode) contains a di-leucine at an identical position with respect to the C-terminus of the protein (34), whilst regions flanking this motif differ substantially from the mammalian homologues. Although the intracellular localization of these MNK homologues is yet to be determined, the broad conservation of this di-leucine suggests that this sequence may function in TGN targeting for each homologue of MNK. The copper-transporting P-type ATPase affected in Wilson disease, WND (ATP7B), is closely related to MNK (35-37), and has been localized to the TGN of hepatomas and hepatocytes (38,39). The WND homologues of human (35-37), mouse (40), rat (41) and sheep (42) each contain three consecutive leucines at a position corresponding to the L1487L1488 motif of MNK homologues within their C-termini. It is possible that these leucine residues function in TGN targeting of WND homologues by a similar mechanism to that used by L1487L1488 in MNK.

The L1487L1488-AA mutation (and the mislocalized truncated forms of MNK) produced a localization in basal media which appeared indistinguishable from that of elevated copper on the MNK(wt) protein. We previously have proposed a model whereby copper may increase exocytic rates of MNK trafficking from the TGN to the plasma membrane (7). However, the apparent similarity of the basal localization of the MNK(L1487L1488-AA) protein to that of the copper-stimulated MNK(wt) protein highlights the possibility that the copper-induced shift in MNK(wt) localization towards the plasma membrane may involve reducing the suggested activity of the di-leucine in retrieving MNK to the TGN. In this model, the net increase in MNK(wt) at the plasma membrane would occur by reducing the trafficking of the protein through the endocytic arm of the recycling pathway. Consistent with this proposal was the observation that stable expression of the L1487L1488-AA mutation in CHO-K1 cells conferred reduced intracellular copper relative to parental CHO-K1 cells, indicating that mutation of the di-leucine did not abrogate the Cu efflux ability of the ATPase. If indeed the di-leucine signal has a role in recycling MNK from the plasma membrane to the TGN, the observation that the L1487L1488-AA mutant protein is able to efflux copper as efficiently as the wild-type protein suggests that the plasma membrane is the site where the protein functions in copper efflux, and that constitutive retrieval to the TGN is not necessary for this efflux function.

In summary, the demonstration that the amino acids L1487L1488 were essential for the predominant TGN localization of MNK identifies an important component of the protein, with a putative role in internalization from the plasma membrane. The recycling pathway followed by MNK involves trafficking of the protein through several membrane compartments. It will be important in future studies to determine whether multiple signals exist to direct MNK trafficking through these various compartments and, if so, whether these signals are influenced by intracellular copper levels. These studies will be essential for understanding how the MNK protein functions in copper transport to copper-dependent enzymes and in the maintenance of intracellular copper homeostasis.

MATERIALS AND METHODS

Affinity-purified MNK antibody

The affinity-purified MNK antibody was raised against the cytoplasmic region of MNK containing the first 590 amino acids which had been expressed in Escherichia coli, and has been described previously (6,7).

Site directed in vitro mutagenesis and transient expression of MNK constructs

The desired mutations were introduced using single-stranded oligonucleotide primers using the Transformer mutagenesis kit (Clonetech). These primers also altered restriction endonuclease sites for identification of mutated plasmids without affecting the encoded amino acid sequence. The template used for mutagenesis reactions was a plasmid containing the last 334 bp of MNK coding sequence derived as a PstI-SmaI subclone of the plasmid pCMB19 which contains the 4.6 kb coding sequence of the MNK cDNA (11,43). The fidelity of mutagenesis reactions and introduction of the desired mutation were confirmed by automated DNA sequencing. After reconstitution of the 5[prime] portion of the MNK cDNA, completed MNK constructs were subcloned into the mammalian expression vector, pCMB43, as described previously (11,43). Transfection of constructs into CHO-K1 cells was performed using the cationic lipid Lipofectamine (Life Technologies) using the manufacturer's suggested protocol. CHO-K1 cells were seeded on glass coverslips 12 h prior to transfection and were ~40% confluent at the time of transfection.

Isolation of stably expressing MNK cells lines

CHO-K1 clones stably expressing either the MNK(wt) or MNK(L1487L1488-AA) protein were isolated using a modified version of the mammalian expression vector, pCMB43. This vector was used previously to generate G418-resistant stable lines expressing MNK(wt) protein (11). However, to increase the frequencies of G418-resistant clones that expressed MNK, we replaced the neoR gene and its SV40 promoter with a promoterless neoR gene immediately following an internal ribosome entry site (IRES), and designated this modified vector, pCMB77. This strategy has been used successfully previously to increase expression frequencies of transfected genes in mammalian cells (44), and expression vectors employing this strategy are commercially available (Clontech). This modification led to the production of a bicistronic mRNA expressed from the CMV promoter, which contained the open reading frame of MNK in the first cistron and the neomycin resistance-coding sequence in the second cistron. With this modified expression vector, CHO-K1 cells expressing either MNK(wt) or MNK(L1487L1488-AA) were isolated by selection for 10 days in G418 (700 µg/ml) after transfection using Lipofectamine. Resistant colonies were screened for MNK expression by indirect immunofluorescence.

Indirect immunofluorescence microscopy

Detection of the MNK protein in CHO-K1 cells was performed as described previously (7) with the following minor modifications. Cells were cultured on glass coverslips in Eagle's media (Trace BioSciences) containing 0.2 mM proline and 10% fetal calf serum (FCS) for the indicated times and fixed by immersion for 30 s in acetone at -20°C. Cells were blocked by incubating overnight in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at 4°C, then incubated for 1 h at room temperature with affinity-purified MNK antibodies diluted in blocking solution (1:50). After washing six times with PBS over 2 h, cells were incubated with affinity-purified fluorescein isothiocyanate (FITC)-conjugated sheep anti-rabbit IgG antibodies (1:300) (Silenus). Cells were washed in PBS overnight and the coverslips were then mounted onto glass slides using 2.6% DABCO [1,4-diazabicyclo-(2.2.2) octane] (Sigma) in 90% glycerol. Images were analysed using a 100× oil objective with a Reichert-Jung Polyvar microscope. The acetone fixation conditions used in combination with the dilution of the affinity-purified MNK antibodies prevented detection of the very low levels of endogenous hamster MNK protein in CHO-K1 cells by immunofluorescence (data not shown).

Preparation of plasma membrane lawns

PM lawns were prepared from cells cultured on glass coverslips coated with poly-l-lysine by high frequency sonication as described previously (7). Plasma membrane fragments were processed for immunofluorescence using the affinity-purified MNK antibodies as described above. Control experiments using antibodies to the plasma membrane marker Na/K ATPase indicated that membranes were derived from the plasma membrane (data not shown).

Western blots

Western blots were carried out as described previously (6) and MNK detected using a chemiluminescence detection kit (Boehringer Mannheim).

Copper accumulation measurements

Copper levels were measured within cells cultured for 20 h in media containing the indicated copper levels and carrier-free 64Cu (Australian Radioisotopes) using an Ultragamma scintillation counter (LKB-Wallac), and standardized against DNA as described previously (6).

ACKNOWLEDGEMENTS

We wish to thank Richard Saffery (Murdoch Institute) for helpful scientific suggestions and Quentin Lang (Department of Genetics) for technical support. This work was supported by grants from the National Health and Medical Research Council to J.C. and J.F.B.M., and from the International Copper Association to J.F.B.M.

ABBREVIATIONS

CHO, Chinese hamster ovary; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; MNK, Menkes disease protein; neoR; neomycin resistance; PBS; phosphate-buffered saline; PM lawn, plasma membrane lawn; TGN, trans-Golgi network, WND, Wilson disease protein.

REFERENCES

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*To whom correspondence should be addressed. Tel: +61 3 9244 7413; Fax: +61 3 9244 7290; Email: jmercer@deakin.edu.au
+These authors contributed equally to this work

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