PAT2 Encodes a Putative AP-3 β Adaptin, a Protein with Conserved Function among Eukaryotes.
(A) Scheme of putative AP-3 β locus and organization. The positions of pat2 alleles (dash arrows), the forward and reverse primers used for RT-PCR (solid arrows), and the point mutation (red letter) are depicted.
(B) Expression of the AP-3 β transcript detected by RT-PCR is shown for pat2-1 (1), the wild type (2), and pat2-2 (3). Tubulin is shown in the bottom panel.
(C) to (E) Complementation test. Localization of PIN1-GFP in wild-type (C), pat2-1 (D), and recovered in pat2-1 transformed with AP-3 β_pro:AP-3 β-GFP (E).
(F) Allelic test showing PIN1-GFP accumulation in pat2-1 pat2-2 F1 seedlings.
(G) to (I) ALP-GFP localization in wild-type yeast (G), alp6Δ mutant (H), and alp6Δ transformed with Arabidopsis AP-3 β (I).
RC, reverse complement. Bars = 10 μm in (C) to (F) and 2 μm in (G) to (I).

Lytic Vacuole Morphology, Biogenesis, and Lytic Degradation Are Impaired in the pat2 Mutant.
(A) and (B) BFA (50 μM, 1 h) treatment on FM4-64 (4 μM) stained control (A) and pat2-1 (B) stele cells. See the complete colocalization of FM4-64 (red) and PIN1-GFP (green) in BFA compartments (yellow) of the control (A). Note that PIN1-GFP aggregations in pat2-1 (green; arrowheads) are distinct from BFA compartments (yellow) (B).
(C) FM4-64 (4 μM, 3.5 h) stains PIN1-GFP–containing compartments in the stele cells of pat2-1 (yellow).
(D) and (E) LysoTracker red (2 μM; 1 h) vacuolar accumulation colocalizes with PIN1-GFP aggregations in pat2-1 roots (yellow) (E) compared with control (D).
(F) and (G) PIP2a-GFP aggregations in pat2-2 get enhanced after 2.5 h of dark treatment (G) compared with light (F).
(H) to (V) pat2 lytic vacuoles show altered morphology. FM4-64 (4 μM; 4 h) staining of the tonoplast in control (H) and pat2-1 (I). The localization of SYP22-YFP in control (J) and pat2-2 (K) epidermal cells. Electron micrographs for control (L), pat2-1 ([M] to [O]), and pat2-2 ([P] to [R]) root endodermal cells. Immunogold labeling shows accumulation of PIN1-GFP on the multilayered endomembranes present in the pat2-1 vacuoles (O). BCECF (10 μM; 40 min) accumulation in control (S) and pat2-1 mutant (T) and LysoTracker red (2 μM; 1 h) in control (U) and pat2-1 (V).
Bars = 5 μm in (A) to (E), 10 μm in (F) to (K), 1 μm in (L) to (R), and 20 μm in (U) and (V).

pat2 Is Not Defective in the Trafficking of Storage Proteins to PSV.
(A) to (G) PSV autofluorescence (imaged immediately after peeling off the seed coat of the dry seeds) in root cells of control (A) and pat2-1 (B). Electron micrographs of control ([C] and [E]) and pat2-1 embryo root cells ([D] and [F]). Histogram evaluating the morphology of PSVs (n = 24 cells) (G).
(H) to (J) Immunogold labeling shows no accumulation of 2S albumin in the intercellular space of pat2-1 (H). Immunoblot (I) and SDS-PAGE (J) reveal no accumulation of 2S albumin and 12S globulin precursors in pat2 mutants, but they accumulate in the vps29, a mutant known to missort the reserve proteins.
Cr, crystalloid; Gl, globoid; Mt, matrix; p2S, 2S precursors; p12S, 12S precursors. Bars = 10 μm in (A) and (B), 5 μm in (C) and (D), 1 μm in (E) and (F), and 0.5 μm in (H).
[See online article for color version of this figure.]

The pat2 Mutant Displays Ectopic Intracellular Protein Accumulation.
(A) to (D) Both PIN1-GFP ([A] and [B]) and aleurain-GFP ([C] and [D]) accumulate intracellularly in pat2-1 (B) or pat2-2 (D) root cells compared with control ([A] and [C]).
(E) Some pat2-1 seedlings arrest on medium without sucrose (arrowhead). Note both the arrested and wild-type-looking seedlings in a population of 7-d-old pat2-1.
(F) and (G) Hypocotyl gravitropic response (n = 75) (F) and lateral root formation (n = 75) (G) are affected in pat2-1.
(H) Intensity of PIN1-GFP signal at the PM of root stele cells is not obviously affected in pat2-1 (n = 110).
Error bars represent sd. Bars = 10 μm in (A) and (B) and 5 μm in (C) and (D).

Ubiquitously Expressed PAT2/AP-3 β Is Localized by a BFA- and Wortmannin-Independent Pathway in Arabidopsis.
(A) to (C) AP-3 β_pro:GUS-GFP expression in embryos ([A] and [B]) and in a 7-d-old seedling (C).
(D) to (F) Pronounced cytosolic localization of AP-3 β-GFP (green) in wild-type root meristematic cells stained shortly by FM4-64 (2 μM) (red) (D). Small AP-3 β-GFP structures can be observed in the epidermal and cortex cells of the upper meristematic zone (arrowhead) (E). AP-3 β-GFP structures look vesiculated in the elongation zone (F).
(G) GFP (green) in 35S_pro:AP-3 β-GFP root cells does not show colocalization with LysoTracker red (2 μM, 1 h) (red).
(H) Treatment of 1 h BFA (50 μM) on AP-3 β_pro:AP-3 β-GFP (green) stained with FM4-64 (4 μM) (red).
(I) to (K) Immunolocalizations show that AP-3 β-GFP structures (green) only randomly associate with SEC21 (I), SYP61 (J), and SYP21 (K). Markers depicted in red.
Bars = 5 μm (D), (E), and (G) to (K) and 2 μm in (F).

pat2 Is Defective in PVC-Dependent Lytic Vacuole Biogenesis.
(A) to (D) Localization of GFP-RABF2b ([A] and [B]) and RABF1-GFP ([C] and [D]) reveals strong vacuolation of the late endosomes/PVCs in pat2-2 ([B] and [D]) compared with the control ([A] and [C]).
(E) to (L) GFP-RABF2b and SYP22-YFP endosome morphology in the control ([E] and [I]) and pat2-2 ([F] and [J]) treated with DMSO and in the control ([G] and [K]) and pat2-2 ([H] and [L]) after 1 h wortmannin (33 μM) treatment. Note the similarity between the wortmannin and pat2 mutation effects on GFP-RABF2 and SYP22-YFP endosomes.
Bars = 10 μm in (A), (B), and (I) to (L) and 20 μm in (C) to (H).

pat2 Is Defective in the Transition between PSV and Lytic Vacuole.
(A) to (H) Live imaging of the PSV autofluorescence in the wild-type ([A], [C], [E], and [G]) and pat2-2 ([B], [D], [F], and [H]) embryos grown on medium without sucrose (long day, 18°C) and imaged immediately after stratification ([A] and [B]), 1 d later ([C] and [D]), 2 d later ([E] and [F]), and 3 d later ([G] and [H]). Note the abnormal development of PSVs in pat2-2 mutant.
(I) to (L) Immunoblot showing the degradation of 2S albumin in control and pat2-1 mutant over 3 d following the germination: immediately after stratification (I), 1 d later (J), 2 d later (K), and 3 d later (L).
Bars = 10 μm in (A) to (F) and 20 μm in (G) and (H).