Characterization of endoplasmic reticulum-localized UDP-D-galactose: Hydroxyproline O-galactosyltransferase using synthetic peptide substrates in arabidopsis

Takuji Oka, Fumie Saito, Yoh ichi Shimma, Takehiko Yoko-o, Yoshiyuki Nomura, Ken Matsuoka, Yoshifumi Jigami

研究成果: Contribution to journalArticle査読

24 被引用数 (Scopus)

抄録

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by high-performance liquid chromatography and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analyses. HGT required a divalent cation of manganese for maximal activity and consumed UDP-D-galactose as a sugar donor. HGT exhibited an optimal pH range of pH 7.0 to 8.0 and an optimal temperature of 35°C. The favorable substrates for the activity seemed to be peptides containing two alternating imino acid residues including at least one acceptor Hyp residue, although a peptide with single Hyp residue without any other imino acids also functioned as a substrate. The results of sucrose density gradient centrifugation revealed that the cellular localization of HGT activity is identical to those of endoplasmic reticulum markers such as Sec61 and Bip, indicating that HGT is predominantly localized to the endoplasmic reticulum. To our knowledge, this is the first characterization of HGT, and the data provide evidence that arabinogalactan biosynthesis occurs in the protein transport pathway.

本文言語英語
ページ(範囲)332-340
ページ数9
ジャーナルPlant physiology
152
1
DOI
出版ステータス出版済み - 1 2010

All Science Journal Classification (ASJC) codes

  • 生理学
  • 遺伝学
  • 植物科学

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