Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

The protein encoded by the Menkes disease gene (MNK) is localised to the Go lgi apparatus and cycles between the trans-Golgi network and the plasma mem brane in cultured cells on addition and removal of copper to the growth med ium, This suggests that MNK protein contains active signals that are involv ed in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membran e, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisati on motifs, using site-directed mutagenesis, protein expression, chemical tr eatment and immunofluorescence. We observed that a di-leucine motif (L1487L 1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane, We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endoc ytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basi s with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.