The Role of Protein Motifs In Membrane Protein Trafficking In S. Cerevisiae

Membrane proteins make up ~30% of the human genome, and G-protein coupled receptors (GPCRs) are the largest family of human membrane proteins, with ~800 members. In recent years, a number of crystal structures of several members of the rhodopsin-like (Class A) GPCRs have been reported, with one agonist-bound active form reported for the adenosine A₂a receptor. However, the path to obtaining crystal structures remains a major challenge, with roadblocks that include heterologous expression, stabilization in membrane-mimetics, and identification of crystallization conditions. Here we address the first hurdle – heterologous expression – in studying human and rat NK₂ receptor expression in Saccharomyces cerevisiae.  Although these two receptors are highly similar in sequence, they display different trafficking profiles and expression yields. Using chimeric receptors, we find that cellular localization appears to be driven by a single domain. Specific trafficking motifs were tested by site-directed mutagenesis, and the effectiveness of these changes were analyzed by confocal microscopy, Western analysis, ligand-binding studies, and examination of cellular stress responses.