SLC-36.1 functions together with PPK-3 in PLR. (A) Images of LAAT-1::GFP– and HIS-24::mCh–positive cell corpses and phagolysosomal vacuoles in slc-36.1(yq110) andppk-3(n2668) single mutants andslc-36.1(yq110);ppk-3(n2668) double mutants. Arrows and arrowheads indicate cell corpses and phagolysosomal vacuoles, respectively. Boxed regions are magnified (2.5×) in insets. Bars, 2 µm. (B) Quantification (mean ± SEM) of the sizes of HIS-24::mCh–positive vacuoles. 15 embryos were scored at the comma stage for each strain. ***, P < 0.001. (C and D) Quantification (mean ± SEM) of the change in phagolysosome sizes (C) and lysosome tubulation events (D). 15 cell corpses were quantified for each strain. ***, P < 0.001. (E) Colocalization of GFP::PPK-3 and mCh::SLC-36.1 to phagosomes in an N2 embryo. Boxed regions are magnified (2.5×) in insets. Bars, 2 µm.(F) Binding curve of Myc–His–PPK-3 to EGFP::SLC-36.1 measured by MST. EGFP was used as the negative control. ΔFnorm, normalized fluorescence ratio of initial fluorescence to fluorescence after laser heating. (G) Purified GST–SLC-36.1(1–53) or GST–SLC-36.1(101–142) was incubated with 35S-labeled PPK-3(1–668 or 669–1497) and pulled down with glutathione-Sepharose beads. Precipitated proteins were resolved by SDS-PAGE and viewed with autoradiography.
