To investigate the function of the protein of interest, lysosomes were stained in the protein of interest overexpressing cells. Neither vesicles nor vacuoles carry lysosomal markers and they were not found to fuse with lysosomes. However, vesicles and vacuoles bind to lysosomes and may interact with them.

Endosomes were visualized by an overexpression of Rab5a-GFP. Vesicles do not carry Rab5a and thereby, are no early endosomes. The vesicles were not found to fuse with endosomes and a binding of endosomes and vesicles was observed rarely.

Figure 1: Vacuoles containing the small isoform do not carry lysosomal markers. A fusion protein of the small isoform and the green fluorescence protein was transfected. The first frame of the 10 min long movie and a temporal and local selection (t = 310 s; 16 x 16 µm) of a vacuole-lysosome interaction are shown.

Figure 2: Vesicles containing the big isoform do not carry lysosomal markers. A fusion protein of the big isoform and the green fluorescence protein was transfected. The first frame of the 10 min long movie and a temporal and local selection (t = 295 s; 9 x 9 µm) of a vesicle-lysosome interaction are shown.

Figure 3: Vesicles containing the big isoform do not carry endosomal markers. A fusion protein of the small isoform and the cherry protein was transfected. The first frame of the 10 min long movie and a temporal and local selection (t = 164 s; 9 x 9 µm) of a vesicle-endosome interaction are shown.