Journal Article

Characterization of an Arabidopsis cDNA Encoding a Subunit of Serine Palmitoyltransferase, the Initial Enzyme in Sphingolipid Biosynthesis

Kentaro Tamura, Naoto Mitsuhashi, Ikuko Hara-Nishimura and Hiroyuki Imai

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 42, issue 11, pages 1274-1281
Published in print November 2001 | ISSN: 0032-0781
Published online November 2001 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pce165
Characterization of an Arabidopsis cDNA Encoding a Subunit of Serine Palmitoyltransferase, the Initial Enzyme in Sphingolipid Biosynthesis

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine in the first step of de novo sphingolipid biosynthesis. In this study, we describe the cloning and functional characterization of a cDNA from Arabidopsis thaliana encoding the LCB2 subunit of SPT. The Arabidopsis LCB2 (AtLCB2) cDNA contains an open reading frame of 1,467 nucleotides, encoding 489 amino acids. The predicted polypeptide contains three transmembrane helices and a highly conserved motif involved in pyridoxal phosphate binding. Expression of this open reading frame in the Saccharomyces cerevisiae mutant strains defective in SPT activity resulted in the expression of a significant level of sphinganine, suggesting that AtLCB2 cDNA encodes SPT. Southern blot analysis and inspection of the complete Arabidopsis genome sequence database suggest that there is a second LCB2-like gene in Arabidopsis. Expression of a green fluorescent protein (GFP) fusion product in suspension-cultured tobacco BY-2 cells showed that AtLCB2 is localized to the endoplasmic reticulum. AtLCB2 cDNA may be used to study how sphingolipid synthesis is regulated in higher plants.

Keywords: Key words: Arabidopsis thaliana — 3-Ketosphinganine — LCB2 — Long-chain base — Serine Palmitoyltransferase (EC 2.3.1.50) — Sphingolipid synthesis.; Abbreviations: DTT, dithiothreitol; ER, endoplasmic reticulum; EST, expressed sequence tag; GFP, green fluorescent protein; LCB, long-chain base; ORF, open reading frame; PMSF, phenylmethylsulfonyl fluoride; RT-PCR, reverse transcription-polymerase chain reaction; SPT, serine palmitoyltransferase; TLC, thin-layer chromatography.

Journal Article.  5603 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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