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.

Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway. A fulllength Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR-based approaches. Arabidopsis SQS is encoded by a small gene family of two genes (SQSl and SQS2) which are organized in a tandem array. SQSl and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity). The isolated cDNA has been assigned to the SQSl gene product, SQSl. RNA blot analysis has shown that the 1.6-kb SQSl mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots. Arabidopsis SQSl isoform is unable to complement the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain. However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 11 1 C-terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302. Labeling studies using ['Hlfarnesyl-P, and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQSl or chimeric Arubidopsis/S. pombe SQS derivatives indicated that the C-terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.Keywords: isoprenoid biosynthesis; Arabidopsis thaliana; yeast; squalene synthase; metabolic channeling.Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (farnesyl-P,) to produce squalene, the first committed precursor for sterol biosynthesis [I]. In plants, as in other eukaryotes, farnesyl-P, lies at a multiple branch point in the isoprenoid biosynthetic pathway [2]. Farnesyl-P, serves as a substrate for the first committed reactions of several branched pathways leading to the synthesis of isoprenoid compounds that are required for plant growth and development, such as phytosterols (membrane structure and function), dolichols (glycoprotein synthesis), ubiquinones (electron transport) or sesquiterpenoid phytoalexins (defense against pathogen attack). Farnesyl-P, also acts as a prenyl donor Enzymes. Squalene synthase, famesyl-diphosphate farnesyl transferase (EC 2.5.1.21); squalene epoxidase (EC 1.14.99.7); lanosterol synthase (EC 5.4.99.7).Note. The Arabidopsis SQS cDNA sequence reported here has been deposited in the GenBank and DDBJ databases under accession number X86692.in protein prenylation, a process that promotes membrane interactions and biological activities of a variety of cell proteins involved in signal transduction, membrane biogenesis and cell growth control [3, 41. In addition to the proposed role of SQS in the control of phytosterol biosynthesis, changes in SQS activity could also affect the flux of isoprenoid compounds down the various branches of t...