David Macar on Arrestin regulation of protein trafficking

David Macar, O'Donnell Lab

Arrestin regulation of protein trafficking: Leveraging evolutionary rate covariation to define a-arrestin-cargo-pairs

The dynamic localization of integral membrane proteins is widely regulated by ubiquitination, which when appended to membrane proteins in response to extracellular signals stimulates their endocytosis and can eventually lead to their trafficking to the vacuole/lysosome. Many membrane proteins lack the binding motifs needed to interact with ubiquitin ligases; Instead, protein trafficking adaptors, such as the a-arrestins, serve as a bridge between the ubiquitin ligase and select membrane cargo proteins to control their protein trafficking fate. What dictates the a-arrestin and membrane protein interaction and what classes of membrane proteins are controlled by each a-arrestin? The answers to these questions is unclear as there are very few known membrane proteins known to be controlled by the a-arrestins. To expand the repertoire of a-arrestins-membrane cargo protein associations, we took a computational approach called Evolutionary Rate Covariation (ERC). ERC compares the rates of evolutionary change of a protein of interest to all the proteins in the proteome across 18 yeast species. Proteins that share a common function, likely share a common selective pressure and therefore will evolve at a similar rate. We performed ERC analyses on each of the yeast a-arrestins and then filtered our data set to focus on transmembrane domain proteins. From this we set an arbitrary cut off of an ERC value of >0.5 and systematically GFP-tagged and assessed the localization of these new putative cargos in wild type cells or cells lacking the a-arrestin of interest. Statistically significant changes in the mean fluorescence intensities, abundance or distribution of GFP-tagged cargos in cells lacking a-arrestins are strong indicators that the protein trafficking of these cargos is a-arrestin-dependent. Using this approach, we have confirmed that 46 new membrane protein cargos are controlled by the a-arrestins. We are currently using bimolecular fluorescence complementation to assess in vivo association of a-arrestins with these cargo proteins. Objectives can be broken into three categories: 1) Identify new cargo-proteins of the yeast a-arrestins. 2) Determine how a-arrestins selectively recognize cargo and define protein-protein interaction motifs between the a-arrestins and cargo. 3) Characterize new trafficking roles of the a-arrestins at other organelles.

Friday, September 21st

12 PM

Langley Hall A219B


21 Sep 2018

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A219B Langley Hall