Figure 5.

PI3P-binding motif of PX domain is essential for DSNX3 function. (A-B) Wing discs are oriented anterior left, dorsal up. (A-A'') UAS-Dsnx3(-RRY)-GFP was expressed in the P compartment of wing discs using en^Gal4. There was no obvious difference in Wg levels between the P and A compartments (arrowheads in A). DSNX3(-RRY)-GFP was distributed throughout the cytoplasm, and no vesicle-like punctate structures can be observed (A''). A'' shows the enlarged view of a segment of the posterior compartment from A'. (B) UAS-Dsnx3(-RRY)-GFP was expressed in the P compartment of Dsnx3 homozygous mutant wing discs using en^Gal4. There was no detectable difference in the Wg levels between A and P compartments, indicating that Wg secretion defect of the Dsnx3 mutant cannot be rescued by the expression of DSNX3(-RRY)-GFP. (C-C''') hWls-V5 and DSNX3(-RRY)-GFP expression vectors were co-transfected into HeLa cells. The subcellular localization of hWls-V5 and DSNX3(-RRY)-GFP proteins were determined by immunostaining. DSNX3(-RRY)-GFP was distributed in the cytoplasm (C, green), and lost the co-localization with hWls-V5 (C', red) and the early endosome marker EEA1 (C'', blue). (D-D''') DSNX3(-RRY)-GFP expression vector was transfected into HeLa cells. DSNX3(-RRY)-GFP protein was detected in the cytoplasm (D, green) and did not co-localized with early endosome marker EEA1 (D', red) and hVps35 (D'', blue).