Identification of tyrosylprotein sulfotransferase in Arabidopsis

Abstract

Tyrosine sulfation is a posttranslational modification common in peptides and proteins synthesized by the secretory pathway in most eukaryotes. In plants, this modification is critical for the biological activities of a subset of peptide hormones such as PSK and PSY1. In animals, tyrosine sulfation is catalyzed by Golgi-localized type II transmembrane proteins called tyrosylprotein sulfotransferases (TPSTs). However, no orthologs of animal TPST genes have been found in plants, suggesting that plants have evolved plant-specific TPSTs structurally distinct from their animal counterparts. To investigate the mechanisms of tyrosine sulfation in plants, we purified TPST activity from microsomal fractions of Arabidopsis MM2d cells, and identified a 62-kDa protein that specifically interacts with the sulfation motif of PSY1 precursor peptide. This protein is a 500-aa type I transmembrane protein that shows no sequence similarity to animal TPSTs. A recombinant version of this protein expressed in yeast catalyzed tyrosine sulfation of both PSY1 and PSK precursor polypeptide in vitro, indicating that the newly identified protein is indeed an Arabidopsis (At)TPST. AtTPST is expressed throughout the plant body, and the highest levels of expression are in the root apical meristem. A loss-of-function mutant of AtTPST displayed a marked dwarf phenotype accompanied by stunted roots, pale green leaves, reduction in higher order veins, early senescence, and a reduced number of flowers and siliques. Our results indicate that plants and animals independently acquired tyrosine sulfation enzymes through convergent evolution.

Fig. 1.

Identification of AtTPST. (A) SDS/PAGE profile of partially purified AtTPST. (B) Photoaffinity labeling of AtTPST by [¹²⁵I]ASA-pPSY1[43-57]. A 62-kDa protein was specifically labeled by the photoaffinity ligand. (C) MALDI TOF-MS spectrum of the tryptic digest of the 62-kDa protein. (D) PMF analysis revealed that 9 MS signals matched 7 internal tryptic fragments of the At1g08030 protein. Matched fragments are underlined. Two fragments show double MS signals due to the partial oxidation of a Met residue. The putative signal peptide is underlined with a dashed line, and the transmembrane domain is boxed. (E) Schematic representation of the alignment between AtTPST and human heparan sulfate 6-O-sulfotransferase 2 (HS6ST2).

Fig. 2.

Tyrosine sulfation activities of recombinant AtTPST expressed in yeast. GST-pPSY1 or GST-pPSK was incubated with [³⁵S]PAPS in the presence of solubilized yeast microsomal fractions expressing AtTPST, or empty vector (control), and analyzed by SDS/PAGE followed by autoradiography.

Fig. 3.

Tissue expression pattern and subcellular localization of AtTPST. (A) Histochemical staining of a 14-day-old Arabidopsis plant transformed with the AtTPSTpro:GUS gene. (Scale bar, 5 mm.) (B) Close-up of RAM in A. (Scale bar, 50 μm.) (C) Subcellular localization of the GFP-AtTPST fusion protein transiently expressed in Arabidopsis protoplasts. (Scale bar, 10 μm.) (D) Effect of BFA treatment (50 μg/mL) on GFP-AtTPST localization. (E) Colocalization of GFP-AtTPST with cis-Golgi marker, mRFP-SYP31. (F) Colocalization of GFP-AtTPST with mRFP-SYP31 after BFA treatment.

Fig. 4.

Phenotypes of a loss-of-function mutant of AtTPST. (A) Schematic map of the T-DNA insertion site of tpst-1 (SALK_009847). (B) Absence of AtTPST transcripts in homozygous tpst-1 plants. RT-PCR analyses were performed on total RNA extracted from tpst-1 or WT plants by using primers specific for AtTPST or for the Arabidopsis ACT3 gene (used as a control). (C) Absence of TPST activities in homozygous tpst-1 plants. GST-pPSY1 was incubated with [³⁵S]PAPS in the presence of solubilized Arabidopsis microsomal fractions derived from tpst-1 or WT plants and analyzed by SDS/PAGE followed by autoradiography. (D) Vertically grown 7-day-old seedlings of tpst-1 and WT. (Scale bar, 5 mm.) (E) Close-up of RAM in D. (Scale bar, 200 μm.) (F) Confocal images of RAM in D stained with propidium iodide. (Scale bar, 200 μm.) (G) Three-week-old plants of tpst-1 and WT grown on B5 agar plates. (Scale bar, 1 cm.) (H) First leaf of tpst-1 and WT cleared by chloral hydrate. In tpst-1 leaves, secondary veins often do not close (indicated by arrows). (Scale bar, 200 μm.) (I) Four-week-old plants of tpst-1, WT, and tpst-1 complemented with AtTPST. (J) Five-week-old plants of tpst-1 and WT.