Understanding Translocation Bottlenecks for Mammalian G-Protein Coupled Receptors Heterologously Expressed in Saccharomyces Cerevisiae

G-protein coupled receptors are 7-transmembrane domain integral membrane proteins that are targets for nearly half of all pharmaceuticals on the market.  Defining the structure of these proteins on an individual basis is of utmost importance for rational drug design, yet structural characterization is generally hindered by low-level expression in native tissues.  In order to facilitate high-resolution structure determination for these proteins, we have achieved high-level expression (~mg/L of culture) for several human GPCRs by expressing them in the yeast Saccharomyces cerevisiae.  However, we have observed that improper translocation into the ER membrane, as evidenced by an intact N-terminal leader sequence fused to expressed receptors, is correlated with intracellular retention and probable misfolding of yeast-expressed human GPCRs.  Examining the mechanism by which translocation fails is critical to understanding and overcoming this bottleneck in yeast, and could lead to re-engineering this host for the robust over-expression of mammalian membrane proteins.      

In this work, we seek to elucidate the limitations for membrane insertion of GPCRs within the yeast ER membrane by creating systematic TM domain truncation mutants for the human adenosine A₂a and A₃ receptors, each with a C-terminal GFP tag.  Trafficking, leader sequence processing, and transmembrane domain topology within the membrane for each receptor truncation mutant were monitored in order to model membrane insertion for the full-length, wild-type protein.  In our system, expression of full-length A₂aR is characterized by complete leader sequence processing and ultimate plasma membrane localization, while full-length A₃R is predominantly intracellular, with the N-terminal leader sequence left intact.  Interestingly, we observe that truncations which include only the N-terminus, first transmembrane domain, and first intracellular loop for both A₂aR and A₃R display a population of mature receptors and a population of pre-Kex2 proteins, indicating that downstream transmembrane domains within each protein influence translocation and leader sequence processing for the full-length protein.  In the case of A₂aR, the addition of subsequent transmembrane domains leading up to the full-length protein was characterized by an increase in mature protein relative to the pre-Kex2 population.  Current studies are focused on determining the orientation of GPCR transmembrane domain segments within the ER membrane through the use of a fluorescence protease protection (FPP) assay, which is applied to yeast cells in vivo.