Plant Molecular Biology 39: 953–967, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis
thaliana cDNA encoding mevalonate diphosphate decarboxylase
, Francis Karst
and Thierry Berg
Laboratoire de G´en´etique Physiologique et Mol´eculaire, ERS CNRS 6099, Institut de Biologie Mol´eculaire et
d’Ing´enierie G´en´etique, 40 Avenue du Recteur Pineau, 86 022 Poitiers C´edex, France (
author for correspon-
INRA, 28 rue de Herrlisheim, 68000 Colmar, France
Received 18 July 1998; accepted in revised form 19 November 1998
Key words: Arabidopsis thaliana, heterologous expression, isoprenoids, mevalonate diphosphate decarboxylase,
sterols, Saccharomyces cerevisiae
Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccha-
romyces cerevisiae identiﬁed an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD
(AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and
isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was
performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs.
The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic
DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous
expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast
PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive
MN19-34 strain deﬁcient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type
sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A
two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer
formation between the plant and yeast heterologous enzymes.
The mevalonate pathway in eukaryotic cells leads to
the synthesis of both sterols and isoprenoids. Sterols
are mainly involved in plasma membrane structure
and are precursors of steroid biosynthesis. Moreover,
in yeast, ergosterol is implicated in the regulation of
the cell cycle . Isoprenoids take part in diverse
cellular functions, such as respiration, protein glyco-
sylation and signal transduction . In higher plants,
this pathway leads also to a large number of plant-
speciﬁc molecules includingphotosyntheticpigments,
growth regulators (e.g. gibberellins and abscisic acid),
phytotoxins, phytoalexins and other plant defence
The nucleotide sequence data reported will appear in the
EMBL Nucleotide Sequence Database under the accession numbers
Y14325A and Y17593.
compounds. A wide variety of secondary metabolites
such as aromatic terpenoids and natural rubbers are
also derived from this pathway [1, 5].
Mevalonate diphosphate decarboxylase (MVD;
EC L-1-1-33) is a key enzyme in the isoprenoid and
sterol biosynthetic pathway. It catalyses the ﬁrst step
of isoprenoid biosynthesis, i.e. the decarboxylation of
the six-carbon mevalonate diphosphate (MVA-PP) to
the ﬁve-carbon isopentenyl diphosphate (IPP). IPP is
the basic skeleton of the biosynthesis of isoprenoids.
The decarboxylation catalysed by MVD is accompa-
nied by the dehydratation of the substrate and the
hydrolysisof one molecule of ATP and requires Mg
In plants, sterols and isoprenoid-derivedmolecules
are localized to various subcellular compartments in
which they fulﬁl their physiological function: sterols