CLONING AND CHARACTERIZATION OF THE ARABIDOPSIS-THALIANA SQS1 GENE ENCODING SQUALENE SYNTHASE - INVOLVEMENT OF THE C-TERMINAL REGION OF THEENZYME IN THE CHANNELING OF SQUALENE THROUGH THE STEROL PATHWAY

Squalene synthase (SQS) catalyzes the first committed step of the ster ol biosynthetic pathway, A full-length Arabidopsis thaliana SQS cDNA h as been isolated by combining library screening and PCR-based approach es. Arabidopsis SQS is encoded by a small gene family of two genes (SQ S1 and SQS2) which are organized in a tandem array. SQS1 and SQS2 have an identical organization with regard to intron positions and exon si zes and encode SQS isoforms showing a high level of sequence conservat ion (79% identity and 88% similarity), The isolated cDNA has been assi gned to the SQS1 gene product, SQS1, RNA blot analysis has shown that the 1.6-kb SQS1 mRNA is detected in all plant tissues analyzed (inflor escenses, leaves, sterns and roots) although the transcript is especia lly abundant in roots. Arabidopsis SQS1 isoform is unable to complemen t the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed y east strain, However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 111 C-ter minal residues of the Schizosaccharomyces pombe enzyme was able to con fer ergosterol prototrophy to strain 5302. Labeling studies using [H-3 ]farnesyl-P-2 and microsomal fractions obtained from yeast strains exp ressing either Arabidopsis SQS1 or chimeric Arabidopsis/S. pombe SQS d erivatives indicated that the C-terminal region of the enzyme is invol ved in the channeling of squalene through the yeast sterol pathway.