title: Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast.
abstract: The limitations to high-level expression of integral membrane proteins are not well understood. The human A(2)a adenosine receptor (A(2)a) and mouse Substance P receptor (SPR) were individually expressed in S. cerevisiae to identify potential cellular bottlenecks for G-protein coupled receptors. In the yeast system, A(2)a was not N-linked glycosylated but was functional and plasma membrane-localized. A(2)a also contained an intramolecular disulfide bond. Substance P receptor was also not N-linked glycosylated in yeast, but, unlike A(2)a, SPR was intracellularly retained, nonfunctional, and did not appear to contain an intramolecular disulfide bond. Since both receptors contain N-linked glycosylation and disulfide bonds in mammalian systems, machinery responsible for interacting with these modifications was investigated-specifically, the potential interactions between the nascent receptor and ER-resident proteins were explored. The chaperones calnexin and protein disulfide isomerase were co-overexpressed with the GPCRs to determine the effect on total and active yields of A(2)a and SPR, as well as on receptor trafficking. The effect of co-expressing the chaperone BiP on the total yields of A(2)a as well as intracellular fates of both receptors were determined. The co-expression of ER resident proteins did not improve A(2)a yields nor did they restore SPR activity or improve SPR cell surface expression. Taken together, these results indicate that an ER-folding bottleneck does not limit the expression of the mammalian receptors in yeast.
authors.affiliation: Department of Chemical Engineering, University of Delaware, 259 Colburn Laboratory, Newark, DE 19716, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Butz
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Niebauer
authors.forename: Anne Skaja
authors.initials: AS
authors.lastname: Robinson
mesh_terms: D000818:Animals; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D006801:Humans; D008322:Mammals; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D043705:Receptor, Adenosine A2A / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; D043705:Receptor, Adenosine A2A; D043562:Receptors, G-Protein-Coupled; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins