Functional Differences between β- and γ-Actin: From Translation Dynamics to Amino Acid Changes

Abstract

The two mammalian cytoplasmic actins - β- and γ- actin - differ only by 4 conservative amino acids at the N-terminus, however their nucleotide coding sequences differ by ∼13% due to silent substitutions. These two actins show major functional differences: gene deletion of β-actin, unlike γ- actin, is embryonic lethal. The exact source of these differences is not well understood. We previously showed introducing 5 point mutations into mouse Actb gene (β-actin) to encode γ-actin protein (Actbc-g) does not affect mouse viability, despite the lack of β-actin protein. This result demonstrates β-actin's requirement for viability depends on its nucleotide, rather than amino acid sequence. Here we investigated the specific role of silent substitutions on β- and γ- actin function. Live cell translation measurements show β-actin translation elongation rate is 1.5-fold faster than γ-actin. Codon-switched constructs show this difference in translation elongation rates, leads to profound effects on cell migration and cell-substrate adhesion. Computational simulations confirmed lower actin synthesis rate near the focal adhesion results in shorter actin bundles. However, at the amino acid level, β- and γ- actin are conserved among all mammals, indicating selection pressure on the 4 conservative substitutions. We show that Actbc-g mice, which lack β-actin protein, show dramatic structural defects in cytoplasmic actin-rich structures, like microvilli in the small intestine and retina. In the retina these defects cause reduced light sensitivity in Actbc-g mice. Phalloidin proteomics of F-actin from Actbc-g tissues reveal altered affinity of specific actin binding proteins (ABPs) for actin in Actbc-g tissues. Together these data suggest actin isoforms’ N-termini co-evolved with ABPs to perform their specific functions in cells. Our results shed light on specific roles of nucleotide and amino acid sequence in actin isoform regulation.