Trafficking of full-length polycystin-2 and polycystin-2 (1–703) with respect to the Golgi apparatus and the trans-Golgi network. (a and j) Immunostaining for acetylated tubulin (red) demonstrates that EGFP fusion proteins with full-length polycystin-2 (PC (fl)) and truncated polycystin-2 (PC2 (1–703)) still enter the primary cilium. (b–e and k–n) LLC-PK1 cells stably synthesizing ECFP fusion proteins with N-acetylgalactosaminyltransferase-2 (T2) and TGN38 (shown in red for better visibility) were transiently transfected with expression plasmids coding for an EGFP fusion protein with the respective polycystin-2 proteins. 3–4 d later, the cells were incubated for 5 h at 15°C before being incubated at 37°C. Immediately before (0 min) and 40 min after (40 min) shifting the cells from 15°C to 37°C, the cells were fixed and visualized in a confocal laser scanning microscope. No overlapping signal can be seen between full-length polycystin-2 and the two marker proteins (b–e), whereas a good overlap was observed for the truncated polycystin-2 protein (k–n). (f–I and o–r) Analogous experiments were performed for the (KFI575-577AAA) mutants of full-length polycystin-2 and polycystin-2 (1–703). For neither mutant protein were we able to detect a colocalization with N-acetylgalactosaminyltransferase-2 and TGN38. Bar, 5 µm.
