Identification and characterization of GONST1, a golgi-localized GDP-mannose transporter in Arabidopsis

Abstract

Transport of nucleotide sugars across the Golgi apparatus membrane is required for the luminal synthesis of a variety of plant cell surface components. We identified an Arabidopsis gene encoding a nucleotide sugar transporter (designated GONST1) that we have shown by transient gene expression to be localized to the Golgi. GONST1 complemented a GDP-mannose transport-defective yeast mutant (vrg4-2), and Golgi-rich vesicles from the complemented strain displayed increased GDP-mannose transport activity. GONST1 promoter::beta-glucuronidase studies suggested that this gene is expressed ubiquitously. The identification of a Golgi-localized nucleotide sugar transporter from plants will allow the study of the importance of this class of proteins in the synthesis of plant cell surface components such as cell wall polysaccharides.

Figure 1.

Sequence of GONST1. (A) Nucleotide sequence of GONST1 cDNA. The two putative N-glycosylation sites are underlined. The GXLNK motif characteristic of GDP-mannose transporters is boxed. (B) Kyte-Doolittle hydrophobicity plot of GONST1 (Kyte and Doolittle, 1982). Potential TMDs are shown with a line above. (C) Alignment with the Golgi GDP-mannose transporters of L. donovani LPG2 and S. cerevisiae Vrg4p. The GXLNK motif characteristic of GDP-mannose transporters is boxed.

Figure 1.

Sequence of GONST1. (A) Nucleotide sequence of GONST1 cDNA. The two putative N-glycosylation sites are underlined. The GXLNK motif characteristic of GDP-mannose transporters is boxed. (B) Kyte-Doolittle hydrophobicity plot of GONST1 (Kyte and Doolittle, 1982). Potential TMDs are shown with a line above. (C) Alignment with the Golgi GDP-mannose transporters of L. donovani LPG2 and S. cerevisiae Vrg4p. The GXLNK motif characteristic of GDP-mannose transporters is boxed.

Figure 2.

RNA Gel Blot Probed with GONST1. (A) Ethidium bromide–stained RNA gel. Lane 1, 2 μg of callus poly(A)⁺ Arabidopsis RNA; lane 2, 2 μg of bolt poly(A)⁺ Arabidopsis RNA. (B) Corresponding RNA gel blot hybridized with digoxigenin-labeled, single-stranded RNA probe of GONST1.

Figure 3.

Functional Complementation of vrg4-2 yeast by pSc-GONST1. Yeast strains JPY25 6c (VRG4), JPY26 3d (vrg4-2), and JPY26 3d transformed with pSc-GONST1 (vrg4-2 + pSc-GONST1) were grown on agar plates, with and without supplements, at 30°C. (A) SC + 0.5 M KCl medium. (B) SC + 0.5 M KCl medium supplemented with 5 mM sodium orthovanadate. (C) SC + 0.5 M KCl medium supplemented with 500 μg/mL hygromycin B.

Figure 4.

The GONST1 Protein Is Expressed in vrg4-2 Yeast Transformed with pSc-GONST1 and Is More Abundant in the Golgi-Rich (P3) Fraction. (A) Protein (5 μg) of the P3 fractions prepared from yeast strains JPY25 6c (VRG4), JPY26 3d (vrg4-2), and JPY26 3d transformed with pSc-GONST1 (vrg4-2 + pSc-GONST1) were resolved by SDS-PAGE and subjected to protein gel blot analysis using the anti-GONST1 antiserum. (B) and (C) The P2 and P3 fractions of JPY26 3d transformed with pSc-GONST1 were resolved and subjected to protein gel blot analysis using the anti-GONST1 antiserum ([B]; 7.5 μg of protein) or antiserum against Anp1p, a yeast Golgi marker protein ([C]; 5 μg of protein).

Figure 5.

Increased GDP-Mannose Uptake in the Golgi-Rich Vesicle Fraction of vrg4-2 Yeast Expressing GONST1. Golgi-rich (P3) vesicles were prepared from yeast strains JPY25 6c (VRG4), JPY26 3d (vrg4-2), and JPY26 3d transformed with pSc-GONST1 (vrg4-2 + pSc-GONST1). Membranes were incubated in reaction mix containing 0.18 μM GDP-¹⁴C-mannose (224.1 mCi/mmol) for 0 or 3 min at 30°C, and reactions were stopped by filtration. GDP-¹⁴C-mannose uptake is calculated as the difference between the two time points. The averages of triplicate assays and standard error bars are shown. The results shown are representative of three independent experiments, and enhanced incorporation of GDP-mannose into the P3 fraction of three other transformants also was observed.

Figure 6.

Distribution of GONST1-YFP in Living Onion Epidermal Cells. (A) Extended focus confocal image through cortical cytoplasm of a single onion cell showing the punctate distribution of fluorescent GONST1-YFP. Bar = 10 μm. (B) Extended focus image through cortical cytoplasm of a single cell as shown in (A) showing clustering of GONST1-YFP fluorescence after BFA treatment. Bar = 10 μm. (C) Extended focus image of a cell as shown in (A). Bar = 25 μm. (D) Image of a single onion cell as in (C) showing colocalization of the actin cytoskeleton (yellow) and GONST1-YFP (green) (left image) and the actin cytoskeleton alone (right image). Bar = 25 μm. (E) Localization of mitochondria (red and right image) and GONST1-YFP (green and left image). Bar = 10 μm.

Figure 7.

Expression of GONST1-GUS in Arabidopsis. (A) Seedling, stained overnight. (B) Inflorescence, stained overnight. (C) Callus, stained for 30 min. Bars = 0.5 cm.