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Fig. 11. A dileucine motif (LL4) is responsible for the internalization observed with the CD8/MNK fusion proteins. The putative internalization motifs in MCF2 (LL1, Y2, LL3, and LL4) were individually mutagenized, resulting in the constructs CD8ALL1, CD8AY2, CD8ALL3, and CD8ALL4, respectively. Plasma membrane staining was seen with CD8-WT (A) and punctate internalization patterns were seen with CD8MCF2 (B), CD8ALL1 (C), CD8AY2 (D), and CD8ALL3 (E), suggesting that each of these motifs were not involved in internalization. However, increased plasma membrane and no punctate staining was observed with CD8ALL4, suggesting that LL4 mediates internalization from the plasma membrane (31).

Fig. 11. A dileucine motif (LL4) is responsible for the internalization observed with the CD8/MNK fusion proteins. The putative internalization motifs in MCF2 (LL1, Y2, LL3, and LL4) were individually mutagenized, resulting in the constructs CD8ALL1, CD8AY2, CD8ALL3, and CD8ALL4, respectively. Plasma membrane staining was seen with CD8-WT (A) and punctate internalization patterns were seen with CD8MCF2 (B), CD8ALL1 (C), CD8AY2 (D), and CD8ALL3 (E), suggesting that each of these motifs were not involved in internalization. However, increased plasma membrane and no punctate staining was observed with CD8ALL4, suggesting that LL4 mediates internalization from the plasma membrane (31).

the plasma membrane (Fig. 10A). However, both CD8MCF1 (Fig. 10B) and CD8MCF2 (Fig. 10C) show intense staining at a pericentriolar complex and punctate staining within the cell. This staining is characteristic of an internalization profile. The similarity of the CD8MCF1 and CD8MCF2 staining patterns imply that the motif responsible for this internalization is contained in both MCF1 and the smaller region MCF2. MCF2 contains four putative internalization motifs—three of the dileucine type (LL1, LL3, and LL4) and one tyrosine motif (Y2) (Fig. 9).

To determine which of these motifs is responsible for the internalization observed, site-directed mutagenesis was used to change each of the motifs. The dileucines were mutated to divalines and the tyrosine to a serine. This resulted in the following constructs: CD8ALL1, CD8AY2, CD8ALL3, and CD8ALL4. These constructs were transfected into human cell lines and the effect of these mutations on internalization monitored (Fig. 11).

Fig. 12. Schematic representation detailing the endocytosis assay used in this assay. (A) At steady-state expression, all potential trafficking routes can be visualized. (B) (i) The CD8-MNK fusion protein trafficks by exocytosis (EX) to the plasma membrane. (ii) The cells in culture are bathed in the antibody OKT8 that recognizes and is allowed to bind to the lumenal domain of CD8. This domain lies exofacial to the cell. (iii) The fusion protein and bound antibody internalize by endocytosis (EN) and this route can be subsequently visualized by indirect immunofluorescence.

Fig. 12. Schematic representation detailing the endocytosis assay used in this assay. (A) At steady-state expression, all potential trafficking routes can be visualized. (B) (i) The CD8-MNK fusion protein trafficks by exocytosis (EX) to the plasma membrane. (ii) The cells in culture are bathed in the antibody OKT8 that recognizes and is allowed to bind to the lumenal domain of CD8. This domain lies exofacial to the cell. (iii) The fusion protein and bound antibody internalize by endocytosis (EN) and this route can be subsequently visualized by indirect immunofluorescence.

The expression patterns of CD8ALL1 (Fig. 11C), CD8AY2 (Fig. 11D), and CD8ALL3 (Fig. 11E) were almost identical to that of CD8MCF2 (Fig. 11B), demonstrating that the internalization profiles observed were independent of LL1, Y2, and LL3. However, the localization of CD8ALL4 (Fig. 11F) was primarily at the plasma membrane and very little, if any, punctate staining was present. This suggests that the dileucine motif, LL4, mediates the internalization of the CD8 fusion proteins. These experiments examine steady-state expression and it is therefore possible that the dileucine motif is important in the trafficking of the fusion protein to the plasma membrane. Therefore, mutation of this signal could prevent trafficking to the plasma membrane and therefore internalization.

To confirm the role of LL4 in internalization from the plasma membrane, an internalization assay was performed. This assay enables only the endocytic route to be observed (Fig. 12). Transient trans-fections of expression constructs CD8-WT (Fig. 13A), CD8MCF1 (Fig. 13B), and CD8LL4 (Fig. 13C) were analysed using this assay. The results clearly demonstrate that the punctate staining observed results from internalization from the plasma membrane and is mediated by LL4.

To determine which organelles in the endocytic pathway are used in this internalization, a series of colocalization experiments with known markers of the endocytic pathway were performed. These included CD63 (late endosomes), Lysotracker (lysosomes), transferrin receptor (recycling endosomes), and TGN46 (trans-Golgi network). Figure 14 compares the internalization profiles of CD8MCF1, the transferrin receptor containing recycling endosomes and TGN46. There is clearly a

Fig. 13. Confirmation that the observed punctate patterning is a result of internalization from the plasma membrane. Expression patterns of CD8-WT, CD8MCF1, and CD8LL4 using the endocytosis assay described in Fig. 12. The CD8-WT signal remains at the plasma membrane (A), whereas punctate staining is observed with both CD8MCF1 (B) and CD8LL4 (C). This patterning arises as a result of internalization from the plasma membrane and confirms the role for LL4 in this internalization (31).

Fig. 13. Confirmation that the observed punctate patterning is a result of internalization from the plasma membrane. Expression patterns of CD8-WT, CD8MCF1, and CD8LL4 using the endocytosis assay described in Fig. 12. The CD8-WT signal remains at the plasma membrane (A), whereas punctate staining is observed with both CD8MCF1 (B) and CD8LL4 (C). This patterning arises as a result of internalization from the plasma membrane and confirms the role for LL4 in this internalization (31).

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