Characterization of Proteins that Interact with the Alpha Subunit of Actin Capping Protein

Actin plays a key role in cell structure, shape, size, and motility. Actin is regulated by a variety of accessory proteins including actin capping protein (CP). CP is a heterodimer composed of an alpha (α) and beta (β) subunit. Lower organisms have one isoform of both the α and β subunit. In contrast, in higher organisms, three α and three β subunit isoforms have been identified. We hypothesize that the three a isoforms have unique cellular and biochemical roles and therefore interact with different cellular proteins. In a previous study, a yeast two hybrid screen was employed using a murine embryonic cDNA library as prey and either αl or α2 as bait to identify protein interactions between αl and α2 and other structural or regulatory proteins. The αl screen identified five interacting clones and the α2 screen identified seven interacting clones. Sequence analysis confirmed the identity of four of the α2 clones as actin, myosin heavy chain 3, serine proteinase inhibitor, and a novel gene on Musculus chromosome 1. The purpose of our research is to characterize the remaining clones. We have isolated total DNA, including genomic and plasmid, from yeast cells and confirmed the integrity of the DNA. The plasmid inserts were amplified via the Polymerase Chain Reaction using vector specific primers to determine insert size. PGR products were sequenced. Preliminary data will be presented