| The limitations to high-level expression of integral membrane proteins are not well understood. The human A2a adenosine receptor (A2a) and mouse Substance P receptor (SPR) were individually expressed in S. cerevisiae to identify potential cellular bottlenecks. In the yeast system, A2a was not glycosylated but was functional and plasma membrane-localized. A2a also contained an intramolecular disulfide bond. SPR was also not glycosylated in yeast, but, unlike A2a, SPR was intracellularly retained, non-functional, and did not contain an intramolecular disulfide bond. Confocal imaging suggests that A2a was not retained at high levels intracellularly, but was quickly turned over from the plasma membrane. SPR also readily passed through the ER and Golgi, but in contrast to A2a, failed to reach the plasma membrane. Since both receptors contain N-linked glycosylation and disulfide bonds in mammalian systems, proteins involved with these modifications were investigated—specifically, the potential interactions between the nascent receptor and ER-resident proteins were explored. The chaperones BiP, calnexin, and PDI were co-overexpressed with the GPCRs to determine the effect on total and active yields of A2a and SPR as well as on receptor trafficking. The co-expression of ER resident proteins did not improve A2a yields, nor did they restore SPR activity or improve SPR cell surface expression. Taken together, these results indicate that the expression of the mammalian receptors in yeast is not limited by an ER bottleneck. It is likely that A2a/SPR self-interaction or interactions with as yet unknown cell proteins control correct folding and assembly. |
