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, 174 (3), 1576-1594

The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit From the Golgi Apparatus

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The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit From the Golgi Apparatus

Myoung Hui Lee et al. Plant Physiol.

Abstract

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis (Arabidopsis thaliana) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4, atpra1.f4, was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na+/K+-ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA:AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus.

Figures

Figure 1.
Figure 1.
atpra1.f4 mutant plants show alterations in development. A, atpra1.f4 plants in soil adopt a smaller stature. B, Shorter root length of atpra1.f4 plants. The root length was measured from wild-type (WT) and atpra1.f4 plants grown on one-half-strength Murashige and Skoog (MS) plates for the indicated time periods. Error bars represent sd (n = 6). Bar = 1 cm. C, Seed yield of atpra1.f4 plants. D, Germination rate of atpra1.f4 plants. Asterisks indicate statistically significant differences at P ≤0.001 (***) between WT and atpra1.f4.
Figure 2.
Figure 2.
HA:AtPRA1.F4 OX and atpra1.f4 mutant plants show hypersensitivity to high concentrations of NaCl and KCl. A to C, Effects of HA:AtPRA1.F4 overexpression and AtPRA1.F4 knockdown mutation on the responses to high concentrations of NaCl and KCl. Plants were grown on one-half-strength MS plates supplemented with or without 100 mm NaCl or 100 mm KCl. Images were taken 29 d after planting. D, Fresh weight of whole plants from 29-d-old plants grown on one-half-strength MS plates supplemented with 100 mm KCl. Three independent experiments were performed on 51 plants. Error bars represent sd (n = 51). Asterisks indicate significant differences at P < 0.01 (**) or P < 0.001 (***) as calculated using Student’s t test. WT, Wild type.
Figure 3.
Figure 3.
HA:AtPRA1.F4 OX and atpra1.f4 plants have lower vacuolar Na+/K+-ATPase and plasma membrane (PM) ATPase activities. A, Effects of HA:AtPRA1.F4 overexpression and AtPRA1.F4 knockdown mutation on vacuolar Na+/K+-ATPase activity. Vacuolar membrane proteins were prepared from isolated vacuoles and used to measure Na+/K+-ATPase activity as described in “Materials and Methods.” Control plants harbor the empty pBIB vector. Asterisks indicate statistically significant differences at P ≤ 0.001 (***) between pBIB and HA:AtPRA1.F4 OX plants and at P ≤ 0.05 (*) between wild-type (WT) and atpra1.f4 plants. Error bars represent sd (n = 3). B, Effects of HA:AtPRA1.F4 overexpression and AtPRA1.F4 knockdown mutation on apoplastic pH. Apoplastic fluid samples were prepared and pH was measured as described in “Materials and Methods.” Error bars represent sd (n = 3). Asterisks indicate statistically significant differences at P ≤ 0.01 (**) between pBIB and HA:AtPRA1.F4 OX plants and between wild-type and atpra1.f4 plants. C, Effects of HA:AtPRA1.F4 overexpression and AtPRA1.F4 knockdown mutation on rhizosphere pH. Ten-day-old seedlings grown on one-half-strength MS plates were incubated in one-half-strength liquid MS medium supplemented with 0.005% Bromocresol Purple for 2 d, and the pH of the incubation medium was measured as described in “Materials and Methods.” Error bars represent sd (n = 3). Asterisks indicate statistically significant differences at P ≤ 0.1 (*) between pBIB and HA:AtPRA1.F4 OX plants and at P ≤ 0.001 (***) between wild-type and atpra1.f4 plants. D, Effects of HA:AtPRA1.F4 overexpression and AtPRA1.F4 knockdown mutation on PM ATPase activity. Inside-out vesicles of PM factions were used to measure PM ATPase activity. Asterisks indicate statistically significant differences at P ≤ 0.001 (***) between pBIB and HA:AtPRA1.F4 OX plants and at P ≤ 0.05 (*) between wild-type and atpra1.f4 plants. Error bars represent sd (n = 3).
Figure 4.
Figure 4.
HA:AtPRA1.F4 localizes to the Golgi apparatus. A to C, Localization of HA:AtPRA1.F4 in HA:AtPRA1.F4 OX plants. Root tissues of HA:AtPRA1.F4 OX plants were fixed and immunostained with anti-HA antibody (A) followed by fluorescein isothiocyanate (FITC)- or tetramethylrhodamine (TRITC)-labeled anti-rat IgG or anti-γ-COP antibody (B) or anti-AtRabF2a antibody (C) followed by TRITC- or FITC-labeled anti-rabbit IgG. To quantify the colocalization in B and C, 30 independent cells were analyzed using the Pearson and Spearman correlation coefficient colocalization plug-in of ImageJ software. The results are shown as scatterplots (right-most images). SE, Stable expression in transgenic plants. Bars = 20 μm. D to F, AtPRA1.F4 localization in protoplasts. HA:AtPRA1.F4 was transformed into protoplasts prepared from transgenic plants expressing ST:GFP or RFP:AtRabF2b, and HA:AtPRA1.F4 localization was examined by immunostaining with anti-HA antibody. ST:GFP and RFP:AtRabF2b were observed directly. TE, Transient expression in protoplasts; WT, wild type. Bars = 10 μm.
Figure 5.
Figure 5.
HA:AtPRA1.F4 overexpression does not inhibit ST:GFP trafficking to the Golgi apparatus. A, Lack of an effect of HA:AtPRA1.F4 on the glycosylation pattern of ST:GFP. ST:GFP was cotransformed with HA:AtPRA1.F4 (10 μg) or R6 (empty vector) into protoplasts prepared from wild-type plants. Protein extracts from protoplasts were treated with Endo H and analyzed by western blotting using anti-GFP and anti-HA antibodies. Gly, Glycosylated form; un-gly, unglycosylated form. B, Trafficking efficiency of ST:GFP to the Golgi apparatus. Trafficking efficiency was determined from the ratio of the Endo H-resistant form versus the total amount of expressed ST:GFP. TE, Transient expression in protoplasts. Error bars represent sd (n = 3).
Figure 6.
Figure 6.
HA:AtPRA1.F4 overexpression and AtPRA1.F4 knockdown inhibit the trafficking of Golgi-dependent vacuolar proteins at the Golgi apparatus. A, Inhibition of vacuolar trafficking in atpra1.f4 plants. Spo:GFP was transformed into protoplasts from wild-type (WT) and atpra1.f4 plants, and their trafficking was examined at various time points by western-blot analysis using anti-GFP antibody. Trafficking efficiency was quantified from the ratio of the processed form versus the total amount of expressed protein. Error bars represent sd (n = 3). Asterisks indicate statistically significant differences at P ≤ 0.05 (*) between wild-type and atpra1.f4 plants. B, Complementation of the defect in vacuolar trafficking of Spo:GFP in atpra1.f4 protoplasts. Spo:GFP was cotransformed with different amounts of HA:AtPRA1.F4 (0.1, 1, and 5 μg) into atpra1.f4 protoplasts, and the trafficking of Spo:GFP was examined at 36 h after transformation by western-blot analysis using anti-GFP antibody. C and D, Inhibition of vacuolar trafficking in HA:AtPRA1.F4 OX plants. Spo:GFP (C) and AALP:GFP (D) were transformed into protoplasts from HA:AtPRA1.F4 OX plants, and their trafficking was examined at various time points by western-blot analysis using anti-GFP antibody. Trafficking efficiency was quantified from the ratio of the processed form versus the total amount of expressed protein. Error bars represent sd (n = 3 in C and n = 2 in D). Asterisks indicate statistically significant differences at P ≤ 0.05 (*) or P ≤ 0.01 (**) between R6 and HA:AtPRA1.F4. TE, Transient expression in protoplasts.
Figure 7.
Figure 7.
HA:AtPRA1.F4 overexpression differentially inhibits the trafficking of TGN-localized SNARE proteins at the Golgi apparatus. A to C, Effects of HA:AtPRA1.F4 overexpression on the localization of SYP41:GFP, SYP51:GFP, and SYP61:GFP. The indicated SNARE and KAM1ΔC:mRFP constructs were transformed into wild-type protoplasts together with HA:AtPRA1.F4 or R6 (empty vector), and the localization of SYP41:GFP (A), SYP51:GFP (B), or SYP61:GFP (C) was examined. For colocalization of SNAREs with KAM1ΔC:mRFP, images (n = 35 and 30 for SYP41, n = 35 and 45 for SYP51, and n = 21 and 30 for SYP61 without and with HA:AtPRA1.F4, respectively) were analyzed using the Pearson and Spearman correlation coefficient colocalization plug-in of ImageJ software. The results are shown as scatterplots (right-most images). Bars = 10 μm. D, Quantification of colocalization. The extent of colocalization of the indicated SNARE proteins with KAM1ΔC:mRFP was calculated and presented in relative values. Error bars represent sd (n = 37 and 53 for SYP41, n = 34 and 41 for SYP51, and n = 19 and 20 for SYP61 without and with HA:AtPRA1.F4, respectively). Asterisks indicate statistically significant differences at P ≤ 0.05 (*) or P ≤ 0.001 (***) between R6 and HA:AtPRA1.F4. TE, Transient expression in protoplasts.
Figure 8.
Figure 8.
HA:AtPRA1.F4 overexpression inhibits the trafficking of PM proteins at the Golgi apparatus. AHA2:GFP was introduced into wild-type protoplasts together with R6 or HA:AtPRA1.F4 (A) or both HA:AtPRA1.F4 and KAM1ΔC:mRFP (B), and the localization of PM proteins was examined. TE, Transient expression in protoplasts. Bars = 10 μm.
Figure 9.
Figure 9.
HA:AtPRA1.F4 overexpression does not inhibit the trafficking of secretory proteins. A and B, HA:AtPRA1.F4 overexpression does not inhibit invertase:GFP secretion. A, Plants harboring invertase:GFP were crossed with HA:AtPRA1.F4 OX plants, and homozygous plants were screened in the F2 population. Localization of invertase:GFP was examined in leaf tissue from homozygous plants expressing both invertase:GFP and HA:AtPRA1.F4. SE, Stable expression in transgenic plants; WT, wild type. B, invertase:GFP was cotransformed into wild-type protoplasts with HA:AtPRA1.F4 or R6, and protein extracts from protoplasts and incubation medium were analyzed by western blotting. C, Protoplasts; M, medium; TE, transient expression in protoplasts. C and D, HA:AtPRA1.F4 overexpression does not inhibit secGFP secretion. C, secGFP was transformed into HA:AtPRA1.F4 OX protoplasts alone or cotransformed into wild-type protoplasts together with HA:AtPRA1.F4 or R6. Subsequently, protein extracts from protoplasts and incubation medium were analyzed at 24 h after transformation by western blotting. Asterisks indicate statistically significant differences at P ≤ 0.001 (***) between R6 and Sar-1[H74L]. D, Protein extracts from protoplasts and incubation medium were prepared at 36 h after transformation, treated with Endo H, and analyzed by western blotting using anti-GFP and anti-HA antibodies. Gly, Glycosylated form; processed, processed form; un-gly, unglycosylated form.
Figure 10.
Figure 10.
HA:AtPRA1.F4 OX plants display reduced vacuolar pH and higher levels of vacuolar ATPase activity. A, Vacuolar pH in root epidermal cells of 6-d-old seedlings was measured as described in “Materials and Methods.” Three independent experiments were performed with more than 50 cells from 15 seedlings in each experiment. The asterisk indicates a significant difference at P ≤ 0.05 (Student’s t test). Error bars represent sd (n = 202 in pBIB and n = 195 in HA:AtPRA1.F4 OX plants). B, Effect of AtPRA1.F4 overexpression on vacuolar ATPase activity. Vacuolar ATPase activity was measured using purified vacuolar proteins prepared from 17-d-old seedlings as described in “Materials and Methods.” Wild-type activity was set to 100%. The asterisk indicates a significant difference at P ≤ 0.05 calculated using Student’s t test. Error bars represent sd (n = 3). C, Effect of ConcA on vacuolar pH. Six-day-old seedlings of control plants and HA:AtPRA1.F4 OX plants were incubated in 1 μm ConcA or dimethyl sulfoxide (DMSO) for 20 h before measurement of pH. Asterisks indicate significant differences at P ≤ 0.05 (*) and P ≤ 0.01 (**) by Student’s t test. Error bars represent sd (n = 3).
Figure 11.
Figure 11.
HA:AtPRA1.F4 OX and atpra1.f4 plants display highly altered Golgi apparatus. A, Golgi morphology. Ultrathin sections of leaf tissues from wild-type (WT), atpra1.f4, and HA:AtPRA1.F4 OX plants were examined by TEM. The inset in the atpra1.f4 image shows a circular form of the Golgi apparatus. Bars = 0.5 μm. B, Quantification of the vesiculated Golgi apparatus. The indicated numbers of Golgi apparatuses were grouped into two categories based on morphology. One group displayed normal morphology (representative image shown at top in A); the other group displayed highly expanded and/or vesiculated structures (representative image shown at bottom in A). Two or more of the Golgi-associated vesicles were larger than 3,000 nm2 (the size of the vesicle) and were considered to be an expanded form in the quantification. C, Effect of HA:AtPRA1.F4 overexpression and AtPRA1.F4 knockdown mutation on vesicle size. The vesicle volume was calculated using ImageJ (values are shown in the box plot). The horizontal line in each box represents the median value of the distribution. The boundaries of the box represent the lower and upper quartile values. The whiskers extending vertically from the bottom and top portions of each box represent the extent of the rest of the data. The number of Golgi apparatuses examined was 81, 85, and 97, respectively, for control, atpra1.f4, and HA:AtPRA1.F4 OX plants.

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