Cbda16

Physiological copper

600|uM

Fig. 5. Mutation of all copper-binding domains abolishes copper-induced relocalization. Each of the cysteine residues in all the six copper-binding domains (CXXC) at the N-terminus of MNK were mutagenised to serines. This mutagenised variant (CBDA1-6), although localized to the TGN, did not move to the plasma membrane on the addition of copper. (Reprinted from ref. 17 with permission from Oxford University Press.)

4.2. Using Reporter Molecules to Identify Intracellular Trafficking Motifs

To understand the copper-induced trafficking of MNK in more detail, studies were initiated into the identification of the motifs/domains involved. In order to identify these signals, we used the reporter molecule CD8. CD8 is a type-1 glycoprotein that is primarily localized to the plasma membrane. For these studies, it can be essentially divided into three domains: (1) lumenal domain; (2) transmembrane domain; (3) cytoplasmic domain (Fig. 7). The N-terminal lumenal domain, which in the wild-type protein sits exofacially to the cell, is recognized by the monoclonal antibody OKT8, which is used to follow the trafficking patterns of the protein within the cell.

The strategy employed involved the excision of either the transmembrane domain or cytoplasmic domain of CD8 and subsequent replacement with the comparative domains from MNK. If any of these newly introduced domains contains signals involved in MNK trafficking, it is likely that these signals will alter the trafficking characteristics of the fusion protein, when compared to the wild-type protein.

4.3. Identification of a Trans-Golgi Network Retention Signal

Many Golgi resident proteins such as TGN38 and ß1,4-galactosyltransferase (29) are retained in the TGN by their transmembrane domain(s). It is therefore feasible that MNK is also retained in a similar manner. To study this, chimeric proteins consisting of the lumenal and cytoplasmic domain of the reporter molecule CD8 fused to different transmembrane domains of the MNK protein were generated. Localization of these chimeric proteins was monitored by indirect immunofluorescence. CD8 wild type (CD8-WT), as expected, localizes primarily to the plasma membrane (Fig. 8B). The insertion of the MNK trans-membrane domain 7 (TM7) into CD8 results in a staining pattern corresponding to the endoplasmic reticulum (Fig. 8H), suggesting that this protein is misfolding and not able to traverse through the secretory pathway. However, on replacing the transmembrane domain of CD8-WT with MNK transmembrane domain 3 (TM3), a staining pattern almost identical to that of the trans-Golgi network resident marker TGN46 is observed (Fig. 8E). The colocalization of these two proteins is confirmed on superimposition of these two images (Fig. 8F). These results suggest

Fig. 6. Only one functional copper domain is required for copper-induced relocalization to the plasma membrane. Six mutants, each containing only one functional copper domain, were generated. All mutants were primarily resident at the TGN at basal copper levels, and all mutants trafficked to the plasma membrane on addition of media containing 600 M of copper. (Reprinted from ref. 17 with permission from Oxford University Press.)

Fig. 6. Only one functional copper domain is required for copper-induced relocalization to the plasma membrane. Six mutants, each containing only one functional copper domain, were generated. All mutants were primarily resident at the TGN at basal copper levels, and all mutants trafficked to the plasma membrane on addition of media containing 600 M of copper. (Reprinted from ref. 17 with permission from Oxford University Press.)

Fig. 7. Schematic representation showing the domains in CD8 used in this study. The N-terminus lumenal domain contains the epitope recognized by the monoclonal antibody OKT8. The transmembrane domain and cytoplasmic domain were excised and replaced with comparative domains from MNK using restriction-enzyme double digests ApaLHSalI and SalI/BamHI, respectively.

Fig. 7. Schematic representation showing the domains in CD8 used in this study. The N-terminus lumenal domain contains the epitope recognized by the monoclonal antibody OKT8. The transmembrane domain and cytoplasmic domain were excised and replaced with comparative domains from MNK using restriction-enzyme double digests ApaLHSalI and SalI/BamHI, respectively.

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