Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting

Abstract

All eukaryotic cells present at the cell surface a specific set of plasma membrane proteins that modulate responses to internal and external cues and whose activity is also regulated by protein degradation. We characterized the lytic vacuole-dependent degradation of membrane proteins in Arabidopsis thaliana by means of in vivo visualization of vacuolar targeting combined with quantitative protein analysis. We show that the vacuolar targeting pathway is used by multiple cargos including PIN-FORMED (PIN) efflux carriers for the phytohormone auxin. In vivo visualization of PIN2 vacuolar targeting revealed its differential degradation in response to environmental signals, such as gravity. In contrast to polar PIN delivery to the basal plasma membrane, which depends on the vesicle trafficking regulator ARF-GEF GNOM, PIN sorting to the lytic vacuolar pathway requires additional brefeldin A-sensitive ARF-GEF activity. Furthermore, we identified putative retromer components SORTING NEXIN1 (SNX1) and VACUOLAR PROTEIN SORTING29 (VPS29) as important factors in this pathway and propose that the retromer complex acts to retrieve PIN proteins from a late/pre-vacuolar compartment back to the recycling pathways. Our data suggest that ARF GEF- and retromer-dependent processes regulate PIN sorting to the vacuole in an antagonistic manner and illustrate instrumentalization of this mechanism for fine-tuning the auxin fluxes during gravitropic response.

Fig. 1.

Visualization of trafficking of plasma membrane proteins to the lytic vacuole. (A) PIN2-GFP localization at the plasma membrane and endocytic intracellular compartments in untreated epidermal root cells. (B and C) Appearance of GFP signal in lytic vacuoles in PIN2-GFP-expressing seedlings after concanamycin A (1 μM for 6 h) treatment (B) or incubation (6 h) in the dark (C). (D) BRI1-GFP-expressing transgenic lines showing BRI1 localization at the plasma membrane and in intracellular structures. (E and F) BRI1-GFP degradation in lytic vacuoles after concanamycin A (E) and dark (F). (G–I) PIP2-GFP distribution in epidermis cells (G) after concanamycin A (H) and dark (I) treatments. (J and K) Appearance of diffuse vacuolar GFP signal (in green) in PIN2-GFP-expressing cells after dark treatment (2 h) identified morphologically in transmission light image (in red) (J) or by endocytic uptake of FM4-64 (K) dye (in red), labeling the tonoplast around the diffuse GFP signal. Arrowheads indicate vacuolar occurrence of GFP signals.

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.

Fig. 3.

Regulation of PIN degradation by plant retromer components. (A and B) Merged images of colocalization (arrowheads) of SNX1-GFP (green) with the PI3P-binding domain FYVE-YFP (red) (A) and retromer component VPS35 (red) (B). (C and D) Two-week-old wild-type seedlings with normal growth on media without sucrose (C) and snx1 mutants arrested in growth (D). (E and F) Rescue of growth defects (E) by transfer on sucrose-containing media (F). (G and H) Substantial vacuolar targeting of the PIN2-GFP in the wild type (G) and even more pronounced in snx1 mutant (H) after dark treatments for 2 h. (I) Reduced PIN2-GFP levels at the plasma membrane in snx1 mutant. Three-dimensional animation of z-stacks (80 μM with 2-μM steps) was obtained. Roots were digitally tilted for outlook at the apical cell surface. False color code was used for PIN2-GFP intensity visualization. (J) Reduced total PIN2 protein levels in snx1 mutants by Western blot analysis. (K and L) Reduced PIN1 protein levels in vps29 (L) compared with wild-type seedlings (K) by z-stack analysis and maximum projection of the PIN1 immunolocalization (≈80 μM, 2-μM steps). (M–P) PIN2 immunolocalization in wild type (M and O) and vps29-3 mutants (N and P). PIN2 accumulation in vps29 was ectopically localized in vacuolar-like structures in meristematic (N) and in elongated root cells (P), suggesting enhanced degradation in vacuoles. Arrowheads depict PIN occurrence in vacuole-like structures. False color code depicts relative fluorescent intensity (I and K–P).

Fig. 4.

Differential PIN targeting to the lytic vacuole during plant development. (A and B) Increased PIN2 targeting to the vacuole in epidermal cells at the upper root side in contrast to the symmetric vacuolar signal in vertically grown roots (A) after dark treatment of gravistimulated (for 3 h) PIN2-GFP roots (B). (C) Differential down-regulation of PIN2-GFP in upper epidermal cells after gravitropic stimulus for 3 h in snx1 mutant seedlings. (D) Enhanced gravity-induced degradation of PIN2 in snx1 mutants revealed by quantitative time course of total PIN2 protein abundance after gravity stimulation. Arrowheads mark differential PIN degradation in lytic vacuoles.