Arabidopsis thaliana contains a single gene encoding squalene synthase
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Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol, brassinosteroid, and triterpene biosynthesis. Arabidopsis thaliana contains two SQS-annotated genomic sequences, At4g34640 (SQS1) and At4g34650 (SQS2), organized in a tandem array. Here we report that the SQS1 gene is widely expressed in all tissues throughout plant development, whereas SQS2 is primarily expressed in the vascular tissue of leaf and cotyledon petioles, and the hypocotyl of seedlings. Neither the complete A. thaliana SQS2 protein nor the chimeric SQS resulting from the replacement of the 69 C-terminal residues of SQS2 by the 111 C-terminal residues of the Schizosaccharomyces pombe SQS were able to confer ergosterol prototrophy to a Saccharomyces cerevisiae erg9 mutant strain lacking SQS activity. A soluble form of SQS2 expressed in Escherichia coli and purified was unable to synthesize SQ from FPP in the presence of NADPH and either Mg2+ or Mn2+. These results demonstrated that SQS2 has no SQS activity, so that SQS1 is the only functional SQS in A. thaliana. Mutational studies revealed that the lack of SQS activity of SQS2 cannot be exclusively attributed to the presence of an unusual Ser replacing the highly conserved Phe at position 287. Expression of green fluorescent protein (GFP)-tagged versions of SQS1 in onion epidermal cells demonstrated that SQS1 is targeted to the endoplasmic reticulum (ER) membrane and that this location is exclusively dependent on the presence of the SQS1 C-terminal hydrophobic trans-membrane domain.
KeywordsErg9 Isoprenoid Mevalonate Plant Squalene Sterol
Cauliflower mosaic virus
Discosoma red fluorescent protein
Green fluorescent protein
3-Hydroxy-3-methylglutaryl coenzyme A
Squalene; SQS, squalene synthase
We wish to thank Francis Karst (INRA, Colmar, France) for yeast strain 2C1 and Paloma Mas (Consorci CSIC-IRTA, Barcelona, Spain) for plasmid pRTL2. This work was supported by grants from the Dirección General de Investigación of the Spanish Ministerio de Educación y Ciencia (BIO2003-1059 and BFU2006-0544 to A. Ferrer and BIO2006-03704 to A. Boronat, all of them including FEDER funds) and from the Direcció General de Recerca de la Generalitat de Catalunya (SGR−00914 to A. Boronat). A. Busquets and V. Keim are recipients of predoctoral fellowships from the Spanish Ministerio de Educación y Ciencia. We gratefully acknowledge the technical support and facilities of the Scientific and Technical Services of the University of Barcelona.
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