Info

Fig. 14. Initial studies demonstrating that internalization of CD8MCF1 occurs via the transferrin receptor containing recycling endosomes. The staining pattern of the internalized CD8MCF1 was compared to that of TGN46 and the transferrin receptor. Although TGN46 (A) and CD8MCF1 (B) are resident in a similar region of the cell, there is no observable overlap between the two proteins (C). The staining patterns of CD8MCF1 (D) and the transferrin receptor containing recycling endosomes (E) are almost identical. This is confirmed on overlapping of the two signals (F) (31).

Fig. 14. Initial studies demonstrating that internalization of CD8MCF1 occurs via the transferrin receptor containing recycling endosomes. The staining pattern of the internalized CD8MCF1 was compared to that of TGN46 and the transferrin receptor. Although TGN46 (A) and CD8MCF1 (B) are resident in a similar region of the cell, there is no observable overlap between the two proteins (C). The staining patterns of CD8MCF1 (D) and the transferrin receptor containing recycling endosomes (E) are almost identical. This is confirmed on overlapping of the two signals (F) (31).

strong overlap with CD8MCF1 and the transferrin receptor (Fig. 14F), although no overlap with TGN46 (Fig. 14C).

To confirm this lack of overlap with TGN46, transfected cells were treated with colchicine. This drug disrupts the pericentriolar complex of the recycling endosomes and also the trans-Golgi network. On addition of colchicine, the accumulation of signal at the center of the cell in the recycling endosomes (Fig. 15B) and the juxtanuclear reticulum of the TGN (Fig. 15E) are no longer visible and the staining is now dispersed within the cell. On superimposition of the images, there is overlap between the dispersed signals of CD8MCF1 and the transferrin receptor (Fig. 15C), but no overlap between CD8MCF1 and TGN46 (Fig. 15F), indicating that CD8MCF1 is internalized through the recycling endosomes.

Although LL4 will internalize CD8 fusion proteins, it is possible that this is an artifactual signal that is activated by the reporter molecule system. To confirm that LL4 is a functional motif in MNK, LL4 was mutated in the full-length MNKMYC cDNA. The expression profile of this mutant protein was then observed. Although, at steady-state expression, MNK is primarily resident at the TGN, previous studies have shown that a small amount of MNK continously recycles between the TGN and plasma membrane. As the mutation of LL4 disrupts the internalization of MNK, there is a gradual accumulation of MNK at the plasma membrane (Fig. 16C,D) compared to the wild-type recombinant protein (Fig. 16A,B). This shows that the dileucine motif LL4 (L1487L1488 in the Menkes amino acid sequence) is essential for internalization from the plasma membrane and has been reported by other groups (32).

These data provide us with two important pieces of trafficking information. First, the dileucine motif L1487L1488 mediates the internalization of MNK from the plasma membrane via the transferrin

Fig. 15. Confirmation that LL4 mediates internalization via the recycling endosomes, but is not sufficient on its own to target the TGN. Transfected cells containing CD8MCF1 were treated with colchicine, which disrupts both the recycling endosomes and the TGN. After treatment with colchicine, significant colocalization is observed with CD8MCF1 (A) and the recycling endosomes (B), shown in (C). However, no colocalization (F) is observed with the disrupted patterns of CD8MCF1 (D) and the TGN (E) (31).

Fig. 15. Confirmation that LL4 mediates internalization via the recycling endosomes, but is not sufficient on its own to target the TGN. Transfected cells containing CD8MCF1 were treated with colchicine, which disrupts both the recycling endosomes and the TGN. After treatment with colchicine, significant colocalization is observed with CD8MCF1 (A) and the recycling endosomes (B), shown in (C). However, no colocalization (F) is observed with the disrupted patterns of CD8MCF1 (D) and the TGN (E) (31).

containing recycling enzymes; second, L1487L1488 is not sufficient on its own to traffick the protein from the plasma membrane to the TGN (12). Therefore, further sorting motifs are required. Current efforts are focusing on identifying these motifs.

0 0

Post a comment