Throughout animal evolution, several innovations allowed organisms to generate genetic ‘novelty’. In vertebrates, repeated genetic duplication events fostered the expansion of several gene families. For example, a family that has three or 4 distinct genes in mammals may only have one equivalent gene in insects. So a recurrent question is: how do insects or other animals that didn't experience these large duplication events generate functional diversity? Nigel Price and colleagues examined this question by looking at the metabolic enzyme carnitine palmitoyltransferase 1 (CPT1) in Drosophila, as most insects only possess one copy of this gene. This enzyme is essential to import long-chain fatty acids into the mitochondria to break them down for energy production. In comparison to flies, mammals express three distinct isoforms in a tissue specific manner, each isoform differing in its affinity for its substrate (carnitine), and its response to its inhibitor (malonyl-CoA). Price and his team specifically hypothesized that Drosophila (and other insects) generated a similar kinetic diversity of this enzyme through alternative splice variants of their single gene. That is, this single ‘insect CPT1 gene’ could give rise to distinct CPT1 proteins through alternative expression of different parts of the gene.

Initially, the team searched genomic databases to identify possible alternative versions of the CPT1 gene in insects. They discovered that most insects possessed a region of their CPT1 gene that could generate two versions of the protein via usage of two interchangeable expressed sequences (exons).

To confirm these predictions, the team undertook gene expression analysis to see whether and how these two putative CPT1 versions were expressed in Drosophila tissues. First, through searches in available collections of expressed genes, they identified two separate CPT1 gene transcripts in Drosophila. These two transcripts only differed by one small portion of the gene where only one or the other functional alternative exon was expressed, the remainder of the gene transcript being identical. The team confirmed the presence of these splice variants in most Drosophila tissues and quantified their relative levels in flight muscle and fat body. Their results established that these two variants were differentially expressed in these tissues indicating possible regulatory and functional differences between the alternative Drosophila CPT1s.

Finally, the team characterized the enzymatic properties of the two splice variants in yeast. They determined that the isoform predominantly expressed in flight muscle (a tissue with high levels of fat oxidation) had overall greater enzymatic activity than the other CPT1 variant, suggesting fine-tuning of the CPT1 enzyme profile according to the metabolic demands of the tissue.

The results of the current study clearly show that flies can still create functional diversity in their metabolic processes despite having comparatively fewer genes than some other animals. In addition, the unique kinetic characterization of a non-mammalian CPT1 enzyme generates interesting hypotheses on the functional diversity in CPT1 across animals. Furthermore, this work begs the question of how functionally different the various CPT1 would be in an animal that experienced repeated duplication events and as a result possesses many additional CPT1 copies.

N. T,
V. N.
K. L.
V. A.
Alternative exon usage in the single carnitine palmitoyltransferase 1 (CPT 1) gene of Drosophila generates functional diversity in the kinetic properties of the enzyme. Differential expression of alternatively spliced variants in Drosophila tissues
J. Biol. Chem.
doi: 10.1074/jbc.M109.072892