Heterologous Expression of the Human D_2S Dopamine Receptor in Protease‐Deficient Saccharomyces cerevisiae Strains

Abstract

The cDNA for the human D,, dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. The original D,, gene and an elongated D,, gene with an Nterminal fusion to the first 24 amino acids of the STE2 gene from S. cerevisiae were introduced into the episomal yeast expression vector YEp51 under the control of the GAL10 promoter. Expression studies performed in a wild-type strain and in two protease-deficient strains of S. cerevisiae revealed that the receptor was functionally expressed with respect to its ligand-binding properties. The K,, values for the binding of the dopamine antagonist [3H]spiperone were calculated to be 1.6 nM for the D,, receptor alone and 1.9 nM for the STE2-D,, chimaera. Both membrane proteins could be further characterized by ligand-displacement studies using certain dopamine agonists and antagonists. D,, dopamine-receptor-specific polyclonal antibodies were used to monitor the heterologous expression of the receptor. Western-blot analysis of membranes prepared from transformed yeast cells producing either the receptor protein alone or the receptor fusion protein revealed apparent molecular masses of 40 kDa (D2s receptor alone) and 42 kDa (STE2/D,, receptor fusion protein). It could be shown that, in comparison to the expression in a wild-type S. cerevisiae strain, the amount of receptor degradation was drastically reduced in the protease-deficient strains. The localizations of the heterologously produced dopamine receptor and of the chimaera in the recombinant yeast were studied by immunogold electron microscopy and were found to be restricted mainly to the vacuole of the cells.

Functional Characterization of an α-Factor-Like Sordaria macrospora Peptide Pheromone and Analysis of Its Interaction with Its Cognate Receptor in Saccharomyces cerevisiae

“…Error bars are given as indicated. (26,41,63,66,75) and for a-factor-like pheromone receptors from basidiomycetes (29,33,57). ␣-factor, which activates the Ste2p receptor in S. cerevisiae, is a 13-amino-acid peptide.…”

Progress toward heterologous expression of active G‐protein‐coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

“…It has been previously observed that heterologous expression of NK 1 , D 2s , and neuromedin U GPCRs in yeast systems led to protein retention within subcellular compartments. [15][16][17] Early time points after induction of expression ($5 h postinduction) were examined to investigate whether the localization in cell populations was different early in expression. As expected, at 5-h postinduction hA 2 aR-GFP appears to uniformly label the plasma membrane [ Fig.…”

“…15 Instead, these receptors are trapped in punctuate structures that are present just below the plasma membrane. 15 Other studies have also cited improper trafficking of recombinant membrane proteins in yeast, 16,17 which suggests that differences between the native mammalian secretory pathway and the yeast secretory pathway can adversely affect the production of proteins in this non-native system. At this point, little information has been published regarding the factors that govern folding and cellular trafficking of heterologously expressed GPCRs in yeast.…”

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

“…To this end, we used BJ3505 and DKY6281 to genetically engineer the tester yeast strains, BJ3505 expressing the neuronal v-SNARE synaptobrevin-2 (BJ3505-Syb2) and DKY6281 expressing the neuronal t-SNAREs syntaxin-1 and SNAP25 (DKY6281-Stx1/S25). Because a subset of mammalian membrane proteins were reported to traffic to the vacuole when ectopically expressed in yeast cells (24), we used the constitutively strong ADH1 promoter to overexpress the neuronal SNARE proteins and examined whether a portion of the proteins were localized to the vacuole. As shown in Fig.…”