Arabidopsis cytosolic Nbp35 homodimer can assemble both [2Fe–2S]
and [4Fe–4S] clusters in two distinct domains
Hirokazu Kohbushi
a
, Yumi Nakai
b
, Shingo Kikuchi
a
, Toshiki Yabe
a,1
, Hiroshi Hori
c
, Masato Nakai
a,
*
a
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita 565-0871, Japan
b
Department of Biochemistry, Osaka Medical College, 2-7 Digaku-cho, Takatsuki 569-8686, Japan
c
Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka 560-8531, Japan
article info
Article history:
Received 19 November 2008
Available online 11 December 2008
Keywords:
Iron–sulfur cluster
Biosynthesis
Molecular scaffold
Cofactor assembly
Arabidopsis thaliana
abstract
Iron–sulfur proteins play physiologically important roles in a variety of metabolic processes in eukary-
otes. In plants, iron–sulfur cluster biosynthesis is known to take place both in mitochondria and chloro-
plasts. However no components that mediate iron–sulfur cluster delivery in the plant cell cytosol have
been identified so far. Here we report identification and characterization of a cytosolic Nbp35 homolog
named AtNbp35 from Arabidopsis thaliana. AtNbp35-deficient Arabidopsis mutants were seedling lethal.
Unlike the previously characterized yeast ScNbp35 which forms a heterotetramer with ScCfd1, AtNbp35
forms a homodimer in the cytosol and can harbor both [4Fe–4S] and [2Fe–2S] clusters on its amino- and
carboxyl-terminal domains, respectively. Taken together, our data suggest that Nbp35 plays a pivotal role
in iron–sulfur cluster assembly and delivery in the plant cell cytosol as a bifunctional molecular scaffold.
Ó 2008 Elsevier Inc. All rights reserved.
Iron–sulfur proteins are defined as proteins in which the iron is
at least partially coordinated by sulfur. Iron–sulfur proteins are
found in all life forms; such as archaebacteria, eubacteria, plants
and also mammals [1]. The iron is either bound to sulfur atoms
of the cysteine residues in the polypeptide backbone or to
inorganic sulfurs. Most frequently, iron–sulfur proteins contain
[2Fe–2S], [3Fe–4S], and [4Fe–4S] clusters. Iron–sulfur proteins
are involved in a variety of metabolic processes including not only
electron transfer reactions but also DNA/RNA metabolisms and
gene regulations. Recent studies have suggested that multiple pro-
tein factors are involved in the process of iron–sulfur cluster bio-
synthesis in the cell [2,3]. In eukaryotic cells, iron–sulfur cluster
biosynthesis primarily takes place in mitochondria where several
so-called ISC components such as Isu1 and Nfs1 have been identi-
fied. Iron–sulfur cluster assembly or delivery outside the mito-
chondria, that is in the cytosol or nucleus, is also known to
require the full set of mitochondrial ISC components [4,5].
Although it remains unclear how iron–sulfur clusters are deliv-
ered from mitochondria to the cytosol, additional cytosolic compo-
nents termed CIA (c
ytosolic iron–sulfur cluster assembly) are
known to be required for iron–sulfur cluster assembly or delivery
in the cytosol [6]. Two homologous P-loop NTPases, namely
Nbp35 and Cfd1, have been identified as essential components of
the CIA from yeast Saccharomyces cerevisiae [7,8] and recently from
mammals [9] and have been shown to form a heterotetramer that
can assemble multiple [4Fe–4S] clusters [10]. In the plant cell,
iron–sulfur cluster biosynthesis is known to take place both
in mitochondria and chloroplasts, where several essential compo-
nents have been identified and characterized extensively [11–
14]. However virtually no components involved in iron–sulfur
cluster delivery or assembly in the plant cell cytosol have been
identified or characterized. Interestingly, Arabidopsis genome
seems to encode one Nbp35 ortholog termed AtNbp35 but no
Cfd1p ortholog. Under this background, we report here that
AtNbp35 is essential for viability of Arabidopsis and form a homodi-
mer in the cytosol which can harbor two different types of iron–
sulfur clusters in the amino- and carboxyl-terminal domains. The
physiological implications of these findings are discussed.
Materials and methods
Strains and growth conditions. The Escherichia coli strains TG-1
and BL21(DE3) were used for plasmid propagation and over-
expression of the recombinant proteins. Arabidopsis (ecotype
Columbia) was grown on MS medium with sucrose or on soil with
a 16-h-light at 23 °C/8-h-dark at 21 °C cycle. T-DNA insertion mu-
tant lines of atnbp35, SALK_056204 and SALK_044980 were pro-
vided by the Salk Institute Genomic Analysis Laboratory. Protein
extracts were prepared from various Arabidopsis tissues and used
for Western analyses as described previously [11]. S. cerevisiae
strains Gal-ScNBP35 and Gal-ScCFD1, in which the NBP35 and the
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.11.138
* Corresponding author. Fax: +81 6 6872 8219.
E-mail address: nakai@protein.osaka-u.ac.jp (M. Nakai).
1
Present address: Institute of Multidisciplinary Research for Advanced Materials,
Tohoku University, Katahira, Sendai 980-8577, Japan.
Biochemical and Biophysical Research Communications 378 (2009) 810–815
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