R. Yelin et S. Schuldiner, Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding, BBA-BIOMEMB, 1510(1-2), 2001, pp. 426-441
A mammalian vesicular neurotransmitter transporter has been expressed in th
e yeast Saccharomyces cerevisiae. The gene encoding the rat vesicular monoa
mine transporter (rVMAT(1)) was cloned in several expression plasmids. The
transporter was expressed at detectable levels only when short sequences us
ing codons favored by S. cerevisiae were fused preceding the start of trans
lation of rVMAT(1). The scarce expression of the wild-type protein was, mos
t likely, due to the fact that part of the N-terminus of the protein is enc
oded by codons not preferred in S. cerevisiae. Furthermore, low growth temp
eratures increased rVMAT(1) expression and altered its processing. Whereas
at 30 degreesC the protein is not glycosylated, at lower temperatures (simi
lar to 16 degreesC) half of the expressed transporters undergo core glycosy
lation. In addition, under these conditions the levels of protein expressio
n significantly increase. Using a functional chimeric protein composed by V
MAT and the green fluorescent protein (GFP), it is shown that the punctate
pattern of intracellular distribution remains invariable at the different t
emperatures. Using a similar fusion sequence, the bovine VMAT isoform 2 (bV
MAT(2)) was also expressed in yeast. The yeast-expressed bVMAT2 binds [H-3]
dihydrotetrabenazine ([H-3]TBZOH) with the same characteristics found in th
e native protein from bovine chromaffin granules. Dodecyl maltoside-solubil
ized bVMAT(2) retains the conformation required for [H-3]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. (C) 2001 Elsevier Science B.V. All rights reserved.