Allelic characterization of the leaf-variegated mutation var2 identiﬁes
the conserved amino acid residues of FtsH that are important
for ATP hydrolysis and proteolysis
*, Eiko Miura
, Yumiko Kaji
, Takashi Okuno
, Masayo Nishizono
and Teru Ogura
Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046,
Japan (*author for correspondence; e-mail firstname.lastname@example.org);
Institute of Molecular Embryology
and Genetics, Kumamoto University, Kuhonji, Kumamoto, Kumamoto 862-0976, Japan
Received 13 August 2004; accepted in revised form 8 October 2004
Key words: Arabidopsis thaliana, chloroplast diﬀerentiation, FtsH, leaf variegation, photosystem II repair
Arabidopsis var1 and var2 mutants exhibit leaf variegation. VAR1 and VAR2 encode similar FtsH
metalloproteases (FtsH5 and FtsH2, respectively). We have previously found many variegated mutants to
be allelic to var2. Each mutant was shown to express a diﬀerent degree of variegation, and the formation of
white sectors was enhanced in severely variegated alleles when these alleles were grown at low temperature.
VAR1/FtsH5 and VAR2/FtsH2 levels were mutually aﬀected even in the weak alleles, conﬁrming our
previous observation that the two proteins form a hetero complex. In this study, the sites of the mutations
in these var2 alleles were determined. We isolated eight point mutations. Five alleles resulted in an amino
acid substitution. Three of the ﬁve amino acid substitutions occurred in Walker A and B motifs of the ATP-
binding site, and one occurred in the central pore motif. These mutations were considered to profoundly
suppress the ATPase and protease activities. In contrast, one mutation was found in a region that contained
no obvious signature motifs, but a neighboring sequence, Gly–Ala–Asp, was highly conserved among the
members of the AAA protein family. Site-directed mutagenesis of the corresponding residue in E. coli FtsH
indeed showed that this residue is necessary for proper ATP hydrolysis and proteolysis. Based on these
results, we propose that the conserved Gly–Ala–Asp motif plays an important role in FtsH activity. Thus,
characterization of the var2 alleles could help to identify the physiologically important domain of FtsH.
Abbreviations: AAA – ATPases associated with diverse cellular activities; EMS – ethylmethane sulfonate;
FtsH – ﬁlamentous temperature sensitive H; IPTG – b-
-thiogalactopyranoside; PSII – photosystem II
The chloroplast is a major site of generating
reactive oxygen species in plant cells, and the
photosynthetic apparatus is constantly damaged
by photooxidation (Asada, 1999; Niyogi, 1999).
To minimize the photodamage, quality control of
chloroplast proteins in which a damaged protein is
rapidly replaced with a newly synthesized protein
is crucial. Recent studies suggest that proteases in
chloroplasts play a key role in the repair cycle of
photodamaged proteins or protein complexes
(Adam and Clarke, 2002).
All chloroplast proteases identiﬁed to date are
of prokaryotic origin (Adam et al., 2001;
Sokolenko et al., 2002). Among these, FtsH is an
Plant Molecular Biology 56: 705–716, 2004.
Ó 2005 Springer. Printed in the Netherlands.