PostDoctoral position: Molecular and functional characterization of
Cd-ATPase transporters from Arabidopsis thaliana
The Labs studies various membrane transporters involved in resistance to
heavy metal in higher plants.
The ABC (ATP-Binding Cassette) proteins are involved in detoxification
of xenobiotics such as herbicides or heavy metals. Our laboratory has
demonstrated the role of this type of proteins in the control of
osmoregulation and ion channels[1,2]. The gene AtMRP2 encoding an ABC
protein (from the MRP subfamily for Multidrug Resistance associated
Protein) implicated in the detoxification of xenobiotics has been cloned
in the Lab[3].
The P-type ATPases constitute another large family of proteins involved
in active transport processes. A part of this family function as
ATP-dependent pumps for the transport of cationic metals through
membranes[4]. This type of proteins has been found in bacteria as well
as mammalians. Although the aminoacid sequences are quite different,
they all present the same topology (eight conserved transmembranous
segments)[5]. These enzymes are implicated in various vital processes
such as secretion, Ca2+-dependent signal transduction. Two homologous
Cu-ATPases are responsibles in the appearance of Menkes[6] (letal early
neuronal degeneration) or Wilson[7] (neuronal degeneration and hepatic
disfunction) diseases due to an hyperaccumulation of the metal in these
organs.
In plants, many proteins of this type have been identified by systematic
sequencing of the genome of agronomic species (Zea mays, Oryza sativa,
Solanum tuberosum or Brassica oleracea) but none has been characterized
today. By analysis of Agrobacterium tumefasciens T-DNA insertional
mutants from Arabidopsis thaliana, a gene encoding a Cd-ATPase has been
identified (hma4). This mutant is defective for this protein (the
insertion is located near the 5=92-end of the gene).
The goal of our project is the functional characterization of Cd-ATPases
from A. thaliana. The complete cDNA of the gene hma4 (3,8 kb) is already
cloned. Now we plane to reintroduce this gene under the control of a 35S
promotor to obtain transgenic plants of A. thaliana overexpressing Hma4.
The metal specificity of the protein will be determined. The affinity
for various cationic metals such as iron, nickel, zinc or radionucleid
as technetium will be probed by : 1) growth of plants in presence of
these metals and accumulation capacities in all the compartments of
plants will be measured ; 2) heterologous expression in Xenopus oocytes
or COS7 cells will allow the measurement of radiolabeled metal flows ;
3) experiences of functional complementation of defective yeast strains
will be realised ; 4) predicted C-term. sequence of Hma4 presents
numerous putative sites of metal attachment. Site directed mutagenesis
of these aminoacids will be considered. Metal transport capacities of
these new mutants will be determined.
The complete sequencing of A. thaliana genome reveals that two
homologous genes (hma2 and hma3) are present. We plane to seek for the
corresponding insertional mutants. The coding sequences of the three
genes present a stretch of few hundred bases highly conserved ; that
could be used to obtain antisense plants defective for the three
Cd-ATPases.
Techniques :
Physiology : measurement of growth and parameters linked to metal
stress (chlorophyll and carotenoid concentration ; biomass) ; metal
content after mineralisation of plants determined by flame atomic
absorption :
Molecular biology : PCR and RT-PCR ; cloning and sequencing ; Southern,
northern and quantitative RT-PCR ; yeast transformation.
Cellular biology : heterologous expression in yeast, oocyte or COS7
cells ; radiolabeled metal flow measurements.
Genetic : A. thaliana plant crossing ; segregation ; obtention of
mutants ; locus transfer in other species.
Please contact :
Dr. Pierre RICHAUD
CEA Cadarache =AD B=E2t. 156
13108 Saint-Paul l=E8s Durance Cedex
00 33 4 42 25 28 97 =AD Fax 00 33 4 42 25 23 64 =AD e-mail
pierre.richaud at cea.fr
Administrative conditions : the postdoctoral position is for one year
and may be prolonged a second year more.
IMPORTANT : If the applicant is unemployed, the PhD must have be
obtained in the last 6 months. However, if in postdoctoral position, the
PhD must have been obtained in the last 2 years.
References
1 Leonhardt N, Marin E, Vavasseur A and Forestier C (1997) Evidence for
the existence of a sulfonylurea-receptor-like protein in plants :
modulation of stomatal movements and guard cell potassium channels by
sulfonylureas and potassium channel openers. Proc. Natl. Acad. Sci. USA
94 : 14156-14161.
2 Leonhardt N, Vavasseur A and Forestier C (1999) ATP binding cassette
modulators control abscisic acid regulated slow anion channels in guard
cells. Plant Cell 11 : 1141-1151.
3 Marin E, Leonhardt N, Vavasseur A and Forestier C (1998) Cloning of
AtMRP1, an Arabidopsis thaliana cDNA encoding a homologue of the
mammalian Multidrug Resistance-associated Protein. Biochim. Biophys.
Acta 1369 : 7-13.
4 Moller JV, Juul B and Le Maire M (1996) Structural organization, ion
transport, and energy transduction of P-type ATPases. Biochim. Biophys.
Acta 1286 : 1-51.
5 Axelsen KB and Palmgren MG (1998) Evolution of substrate specificities
in the P-type ATPase superfamily. J. Mol. Evol. 46 : 84-101.
6 Vulpe C, Levinson B, Whitney S, Packman S and Gitschier J (1993)
Isolation of a candidate gene for Menkes disease and evidence that it
encodes a copper-transporting ATPase. Nat. Genet. 1 : 7-13.
7 Tanzi RE, Petrukhin K, Chernov I, Pellequer JL, Wasco W, Ross B,
Romano DM, Parano E, Pavone L and Brzustowicz LM (1993) The Wilson
disease gene is a copper transporting ATPase with homology to the Menkes
disease gene. Nat. Genet. 4 : 344-350.
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