Butz James A, Niebauer Ronald T, Robinson Anne Skaja
Department of Chemical Engineering, University of Delaware, 259 Colburn Laboratory, Newark, DE 19716, USA.
Biotechnol Bioeng. 2003 Nov 5;84(3):292-304. doi: 10.1002/bit.10771.
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.
人们对整合膜蛋白高水平表达的限制尚未完全了解。人A(2)a腺苷受体（A(2)a）和小鼠P物质受体（SPR）分别在酿酒酵母中表达，以确定G蛋白偶联受体潜在的细胞瓶颈。在酵母系统中，A(2)a未进行N-糖基化，但具有功能且定位于质膜。A(2)a还含有一个分子内二硫键。P物质受体在酵母中也未进行N-糖基化，但与A(2)a不同的是，SPR被保留在细胞内，无功能，且似乎不含有分子内二硫键。由于这两种受体在哺乳动物系统中都含有N-糖基化和二硫键，因此研究了负责与这些修饰相互作用的机制——具体而言，探索了新生受体与内质网驻留蛋白之间的潜在相互作用。伴侣蛋白钙连蛋白和蛋白质二硫键异构酶与GPCR共同过表达，以确定对A(2)a和SPR的总产量和活性产量以及受体转运的影响。确定了共表达伴侣蛋白BiP对A(2)a总产量以及两种受体细胞内命运的影响。内质网驻留蛋白的共表达既没有提高A(2)a的产量，也没有恢复SPR的活性或改善SPR在细胞表面的表达。综上所述，这些结果表明内质网折叠瓶颈并不限制哺乳动物受体在酵母中的表达。
