Fig. 7: PRAFs and GNOM are required for endomembrane trafficking.

a Localization of PIN3-GFP (green) in the designated genetic backgrounds. White dashed arrows indicate uneven distribution of PIN3 at the plasma membrane. Yellow arrowheads mark abnormal aggregations of PIN3 in the cytoplasm. Three independent experiments were performed. b FM4-64 (red) dye distribution (8 μM, 40 min) in stomatal lineage cells of WT, praf4t;5c;8c;9c, and gnom (segregated from gnom^B/E) to compare with WT treated with BFA (70 μM, 60 min). White arrowheads indicate internalized FM4-64 in WT. Yellow arrowheads indicate abnormal FM4-64 aggregations in praf4t;5c;8c;9c, gnom, or in WT treated with BFA. Data represent results of three independent experiments. c Quantification of endocytosis rates based on FM4-64 internalization. Box plots show numbers of FM4-64 positive endosomes per cell in WT, praf4t;5c;8c;9c and gnom after 5, 10, 15, 30, and 40 min of 8 μM FM4-64 treatment, respectively. Box plots show first and third quartile (box), median (line) and mean (cross). n, # cells measured. Student’s unpaired t tests were used to compare with the wild type. Two-sided P values are 0.0001 (for WT vs. praf4t;5c;8c;9c) and 0.0004 (for WT vs. gnom) with 30-min FM4-64 treatment. All other two-sided P values are <0.0001. ***P < 0.001; ****P < 0.0001. d Quantification of sizes of FM4-64-positive compartments in designated backgrounds. Box plots show first and third quartile (box), median (line) and mean (cross). n, # FM4-64 positive compartments measured. Student’s unpaired t tests were used in (c) and (d) to compare with the wild type. All two-sided P values are <0.0001. ****P < 0.0001. e Localization of RabC1 (green, left) and RabE1d (green, right) in designated backgrounds. Red arrowheads mark more diffused RabC1 in the mutants. Cyan arrowheads mark filamentous distribution of RabE1d. Three independent experiments were performed. Scale bars in (a), (b), and (e) are as indicated (μm). f Quantification of vesicular sizes for RabA1e, RabC1, RabD1, RabD2a, RabE1d, and RabF2b in WT, praf4t;5c;8c;9c, and gnom, respectively. Box plots show first and third quartile (box), median (line) and mean (cross). n, # vesicles measured. Student’s unpaired t tests were used. Two-sided P values are 0.0053 (for RabC1 vs. RabC1;gnom) and 0.0112 (for RabF2b vs. RabF2b;praf4t;5c;8c;9c). All other two-sided P values are <0.0001. *P < 0.05; **P < 0.005; ****P < 0.0001. g Proposed working models for the subcellular localization of PRAF8 proteins (left) and trafficking pathways possibly interfered by the absence of the four PRAF genes (PRAF4, PRAF5, PRAF8 and PRAF9) or GNOM (right). In wild-type plants, the polarization of BASL protein (green) in the stomatal lineage cell requires the physical partner, four PRAF proteins (orange), as well as the Golgi-localized Arf GEF GNOM (blue). The PRAF8 proteins are predominantly localized to the plasma membrane, where they may polarize together with BASL. The PRAF8 proteins may also partially associate with the Golgi, TGN/EE and a subset of endosomes/vesicles decorated by RabC1 and RabF2b. Furthermore, the PRAF proteins physically interact with GNOM, possibly leading to the association of GNOM to the RabC1- and RabE1d-decorated membrane structures. In the absence of the four PRAF genes or GNOM, multiple endomembrane markers are similarly defective in morphology and/or distribution (red crosses), suggesting that multiple routes, including endocytosis, secretion, and recycling etc., are commonly disturbed in praf and gnom mutants. We propose that the connected function of PRAF and GNOM plays important roles in endomembrane trafficking and is required for the establishment of BASL polarization in the stomatal lineage cells. PM plasma membrane, NE nuclear envelope, ER endomembrane reticulum, G Golgi, TGN/EE trans-Golgi network/early endosome, SV secretory vesicle, RE recycling endosome, MVB multivesicular body, LV lytic vacuole.