The moonlighting role of pyruvate carboxylase

Moonlighting proteins: old proteins learning new tricks
Moonlighting function of proteins: new roles of proteins which have been discovered for other functions. Here the pyruvate carboxylase is known as converting pyruvate into phosphoenolpyruvate (PEP) in gluconeogenesis, its moonlighting role is involved in the assembly of yeast peroxisomal oligomeric alcohol oxidase (AO).
The discovery of its new role:
1. Ozimek et al. (2003) isolated the yeast mutants that have accumulation of inactive alcohol oxidase monomers (which should be active in octomer). Mutant strains were transformed with a yeast genomic library, fragments that could complement the abnormal phenotype show high similarity with pyruvate carboxylase sequences.
2. Ozimek et al. (2007) identified the region of pyruvate carboxylase gene that are responsible for its moonlighting function by producing the random mutants in the pyruvate carboxylase gene.
3. Hubert et al. (2010) identified the amino sites that are involved in its moonlighting function via PCR based site directed mutagenesis. And multiple sequence alignments showed all these sequences are conserved in the normal yeast strains but the mutant strains with failure alcohol oxidase function.

This serial papers also showed a general idea for the identification of new proteins and the coding genes:
Mutant discovered -->
Transformed with whole genomic library to find out the genes that could compliment the mutant -->
Database search and multiple line alignments to see what these genes are, or what they are similar with -->
Randomly disrupt the gene to see which part is critical for the function -->
Site directly mutagenesis, which will restrict the essential sites within the gene or the DNA sites within the gene.