Fig. 2.

Cellular and molecular requirements of the PIN trafficking to lytic vacuoles. (A and B) Latrunculin B (LatB; 20 μM) treatment (B) compared with control (A) revealing actin-dependent trafficking of the PIN2-GFP to the vacuole as visualized by dark treatment (2 h). (C and D) Dark treatments (2 h) showing the reduction of PIN2-EosFP in vacuolar targeting at 25 μM BFA (C) and a complete block at 50 μM BFA (D). (E) In the transgene carrying the engineered BFA-resistant version of GNOM ARF GEF (GNOM^ML), the vacuolar trafficking of PIN2-EosFP is still BFA-sensitive. (F–J) ARF GEF-dependent FM4-64 uptake (2 h) to the tonoplast (indicated by arrowheads) of untreated cells (F) and cells treated with 25 μM BFA (G), 50 μM BFA (H), and 50 μM BFA in the BFA-resistant version of GNOM^ML (I) and after heat-shock induction (37 °C for 2 h) of the constitutively active ARF1^QL (J). (Inset) FM4-64 and ARF1^QL-YFP colocalization. (K and L) Concanamycin A (1 μM, 6 h)-visualized trafficking of PIN2-GFP (K) and PIP2-GFP (L) to vacuoles inhibited by wortmannin (15 μM, 6 h) (K′ and L′) treatment. (M) Occasional PIN2 localization at the tonoplast after wortmannin (15 μM for 3 h in the dark) treatment. (N) PIN2 protein-stabilizing effect of wortmannin (WM) brefeldin A (BFA), and latrunculin B (LatB) by Western blot analysis. Concomitant drug treatment with protein biosynthesis inhibitor cycloheximide (CHX) was done for inhibition of the PIN2 secretion. (O) Schematic representation of the ARF-GEF (BFA) and PI3K (WM)-sensitive sorting events of PIN2 to the lytic vacuole for degradation. Arrows mark BFA-induced accumulation, and arrowheads mark the PIN2 occurrence in vacuoles or endocytic mistargeting.