Figure 7. The TRPP3 coiled-coil domain trimer is essential for the surface expression of the PKD1L3/TRPP3 complex.

(a) Bar graph comparing acid-induced whole-cell currents from oocytes expressing the indicated constructs. (b) TIRF images of both EGFP and mCherry fluorescence from oocytes expressing the indicated constructs. The images for both combinations were obtained from oocytes injected with the same amount of cRNAs and on the same day after injection, and are representative of >6 different oocytes. Scale bar, 2 μm. (c) Western blot following SDS-PAGE showing TRPP3 and PKD1L3 expressed on the plasma membrane (upper gels) and in the lysate (lower gels) of oocytes expressing the indicated constructs. The actin control shows that only surface proteins were biotinylated. Monomeric and oligomeric TRPP3 are indicated by arrows and asterisks. PKD1L3 showed a full-length band (>250 kDa, arrow heads) and a cleaved band (~180 kDa, open circles) (lower gel, lanes 3, 4, 6 and 7). Disruption of the TRPP3 coiled-coil domain trimer reduced the surface expression of TRPP3 (upper gel, lanes 5–7) and PKD1L3 (upper gel, lanes 6 and 7). Bar graphs at the bottom show the normalized intensity of surface TRPP3 or PKD1L3 proteins when PKD1L3 was coexpressed with WT or the indicated mutant TRPP3. Data were obtained from lines 4, 6 and 7 on the top two gels. The sum of the intensity of all monomeric and oligomeric bands for each protein was used in the calculation. (d), Western blot following SDS-PAGE showing that PKD1L3 (HA-tagged) can be co-immunoprecipitated with all indicated TRPP3 constructs (FLAG-tagged). Monomeric and oligomeric TRPP3 are indicated by arrows and asterisks. Full-length and cleaved PKD1L3 are indicated by arrow heads and open circles. Bar graph at the bottom shows the normalized ratio of the intensity of HA-PKD1L3/FLAG-TRPP3 (WT or the indicated mutants).

(Yang)