Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding
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- 作者：Yelin ; Rodrigo ; Schuldiner ; Shimon
- 关键词：Neurotransmitter transport ; Codon usage ; Heterologous expression ; Protein folding ; Tetrabenazine ; Vesicular monoamine transporter ; Chaperone ; 5-HT ; serotonin ; ADH ; alcohol dehydrogenase ; HA ; hemagglutinin ; MPP+ ; N-methyl-4-phenylpyridinium ; PAG
- 刊名：Biochimica et Biophysica Acta (BBA)/Biomembranes
- 出版年：2001
- 出版时间：February 9, 2001
- 年：2001
- 卷：1510
- 期：1-2
- 页码：426-441
- 全文大小：4.33 M
文摘
A mammalian vesicular neurotransmitter transporter has been expressed in the yeast Saccharomyces cerevisiae. The gene encoding the rat vesicular monoamine transporter (rVMAT1) was cloned in several expression plasmids. The transporter was expressed at detectable levels only when short sequences using codons favored by S. cerevisiae were fused preceding the start of translation of rVMAT1. The scarce expression of the wild-type protein was, most likely, due to the fact that part of the N-terminus of the protein is encoded by codons not preferred in S. cerevisiae. Furthermore, low growth temperatures increased rVMAT1 expression and altered its processing. Whereas at 30°C the protein is not glycosylated, at lower temperatures (
16°C) half of the expressed transporters undergo core glycosylation. In addition, under these conditions the levels of protein expression significantly increase. Using a functional chimeric protein composed by VMAT and the green fluorescent protein (GFP), it is shown that the punctate pattern of intracellular distribution remains invariable at the different temperatures. Using a similar fusion sequence, the bovine VMAT isoform 2 (bVMAT2) was also expressed in yeast. The yeast-expressed bVMAT2 binds [3H]dihydrotetrabenazine ([3H]TBZOH) with the same characteristics found in the native protein from bovine chromaffin granules. Dodecyl maltoside-solubilized bVMAT2 retains the conformation required for [3H]TBZOH binding. We exploited the robust binding to follow the transporter during purification assays on a Ni2+-chelating column. In this report we describe for the first time the heterologous expression of a neurotransmitter transporter in the yeast S. cerevisiae.
16°C) half of the expressed transporters undergo core glycosylation. In addition, under these conditions the levels of protein expression significantly increase. Using a functional chimeric protein composed by VMAT and the green fluorescent protein (GFP), it is shown that the punctate pattern of intracellular distribution remains invariable at the different temperatures. Using a similar fusion sequence, the bovine VMAT isoform 2 (bVMAT2) was also expressed in yeast. The yeast-expressed bVMAT2 binds [3H]dihydrotetrabenazine ([3H]TBZOH) with the same characteristics found in the native protein from bovine chromaffin granules. Dodecyl maltoside-solubilized bVMAT2 retains the conformation required for [3H]TBZOH binding. We exploited the robust binding to follow the transporter during purification assays on a Ni2+-chelating column. In this report we describe for the first time the heterologous expression of a neurotransmitter transporter in the yeast S. cerevisiae.