α‐Arrestin regulation of protein trafficking: Using evolutionary rate covariation to define protein trafficking regulatory networks

Abstract

α-Arrestins, a novel class of protein trafficking adaptors, help cells survive environmental changes by controlling membrane protein trafficking. One hurdle to understanding α-arrestins is that few α-arrestin-cargo pairs have been identified. It is technically challenging to identify membrane cargos trafficked by a-arrestins due to their transient associations and their biochemical nature. To identify α-arrestin-regulated cargos, we used Evolutionary Rate Covariation (ERC), which employs sequence-based signatures to identify genes with similar evolutionary histories. We compared ERC values for α-arrestins with proteins across 18 yeast species. Among the top co-evolving proteins were those previously defined as α-arrestin cargos. We are determining if membrane proteins, not yet identified as α-arrestin cargos, with the highest ERC values (>0.5), interact with α-arrestins by assessing their localization and relative protein abundances in wild-type cells versus those lacking the specific α-arrestins. Fluorescence intensity was used to quantify the abundance and/or subcellular distribution of GFP tagged proteins. Statistically significant changes in GFP abundance or localization between wild-type cells and those lacking α-arrestins demonstrate dependence on these protein trafficking adaptors. This makes them good candidates as new α-arrestin-dependent cargos, which we are confirming by co-localization, bimolecular fluorescence complementation, and biochemical approaches. Using ERC and our fluorescence imaging pipeline, we have quantitatively confirmed that 36 integral membrane proteins, previously unassociated with α-arrestins, display α-arrestin-dependent localization changes. This greatly expands the repertoire of α-arrestin cargo within cells, and raises important functional implications for this family. In conclusion, the ERC approach is a powerful new tool that is able to define protein trafficking regulatory networks, which will undoubtedly be of interest to the cell biology community.