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Genetic Analysis Reveals Domain Interactions of Arabidopsis
Hsp100/ClpB and Cooperation with the Small Heat Shock
Protein Chaperone System
W
Ung Lee,
a
Chris Wie,
a
Mindy Escobar,
a
Ben Williams,
a
Suk-Whan Hong,
b
and Elizabeth Vierling
a
,
1
a
Department of Biochemistry and Molecular Bioph ysics, Uni versit y of Arizona, Tucson, Ari zona 85721
b
Department of Appli ed Plant Sc ienc es, Agricultural Plant Stress Research Center,
Chonnam National Universi ty, Kwang Ju, 500-757, South Korea
We have defined amino acids important for function of the Arabidopsis thaliana Hsp100/ClpB chaperone (AtHsp101) in
acquired thermotolerance by isolating recessive, loss-of-function mutations and a novel semidominant, gain-of-function
allele [hot1-4 (A499T)]. The hot1-4 allele is unusual in that it not only fails to develop thermotolerance to 458C after
acclimation at 388C, but also is sensitive to 388C, which is a permissive temperature for wild-type and loss-of-function
mutants. hot1-4 lies between nucleotide binding domain 1 (NBD1) and NBD2 in a coiled-coil domain that is characteristic of
the Hsp100/ClpB proteins. We then isolated two classes of intragenic suppressor mutations of hot1-4: loss-of-function
mutations (Class 1) that eliminated the 388C sensitivity, but did not restore thermotolerance function to hot1-4, and Class 2
suppressors that restored acquired thermotolerance function to hot1-4. Location of the hot1-4 Class 2 suppressors
supports a functional link between the coiled-coil domain and both NBD1 and the axial channel of the Hsp100/ClpB
hexamer. In addition, the strongest Class 2 suppressors restored solubility of aggregated small heat shock proteins (sHsps)
after heat stress, revealing genetic interaction of the Hsp100/ClpB and sHsp chaperone systems. These results also
demonstrate that quantitative phenotypes can be used for in vivo genetic dissection of protein mechanism in Arabidopsis.
INTRODUCTION
Hsp100/ClpB chaperones are hexameric members of the AAAþ
family of proteins, which are ATPases that couple ATP binding
and hydrolysis to a variety of protein-remodeling activities
(Neuwald et al., 1999; Vale, 2000; Ogura and Wilkinson, 2001;
Lupas and Martin, 2002). Hsp100/ClpB proteins have been
shown to be essential for the development of thermotolerance
to high temperature in bacteria (Goloubinoff et al., 1999), yeast
(Glover and Lindquist, 1998), some parasitic protozoa, and
higher plants (Hong and Vierling, 2000, 2001), which are the
only higher eukaryotes known to express the Hsp100/ClpB
subgroup of chaperones. The demonstration that the highly
heat-induced Hsp100/ClpB protein in Arabidopsis thaliana,
AtHsp101, was essential for acquisition of tolerance to high
temperature also represented a direct link of a heat shock protein
to heat tolerance in plants. Both in vivo and in vitro evidence
indicates that the protective function of these chaperones is
a result of their ability to resolubilize protein aggregates in co-
operation with the Hsp70/DnaK chaperone system (Goloubinoff
et al., 1999; Zolkiewiski, 1999). It remains unknown, however,
whether general removal of aggregated proteins or rescue of
certain critical substrates defines the essential activity of
Hsp100/ClpB in thermotol erance.
This unique subgroup of ATPases contains two AAA modules,
each comprised of a nucleotide binding domain (NBD1 or 2) with
conserved Walker A, Walker B, and sensor motifs and a smaller
C-terminal domain. The AAA modules are flanked by additional
N- and C-terminal domains and are separated by a large coiled-
coil domain, which is actually an insertion into the C-terminal
small domain of AAA module 1 (see Figure 1A). The length of the
coiled-coil domain is a major feature that distinguishes Hsp100/
ClpB proteins from other Clp proteins with two AAA modules
(e.g., ClpA) (Schirmer et al., 1996; Celerin et al., 1998; Nieto-
Sotelo et al., 1999). The recent 3.0-A
˚
structure of a complete
Thermus thermophilus ClpB subunit, and a cryoelectron micros-
copy reconstruction of the hexamer (21-A
˚
resolution), has de-
fined the structure of the different ClpB domains and how they
are oriented relative to each other (Lee et al., 2003). In each AAA
module, NBD1 and 2 have a RecA-like mononucleotide binding
fold, and the C-terminal smaller domain has a mixed a/b
structure. The two AAA modules are stacked on each other
within a subunit such that the hexamer appears as two rings of
six ATPase sites, surrounding a pore or axial channel on the order
of
;16 A
˚
in diameter. The coiled-coil domain, which is inserted
into the smaller domain of NBD1, is 85-A
˚
long and resembles
a two-bladed propeller ringing the outside of the hexamer. Based
on the observation of three different conformations in the crystal
structure, the N-terminal domain and coiled-coil domain are
1
To whom correspondence should be addressed. E-mail vierling@
email.arizona.edu; fax 520-621-3709.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Elizabeth Vierling
(vierling@email.arizona.edu).
W
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.104.027540.
The Plant Cell, Vol. 17, 559–571, February 2005, www.plantcell.org ª 2005 Ameri can Society of Plant Biologists
proposed to be mobile. This is consistent with the fact that
deletion of either domain does not disrupt the hexamer (Mogk
et al., 2003b).
Biochemical studies using both in vitro assays for ATP hydro-
lysis and protein disaggregation (chaperone activity), along with
in vivo assays for thermotolerance, have also begun to dissect
functional domains of Hsp100/ClpB (Smith et al., 1999; Barnett
et al., 2000; Clarke and Eriksson, 2000; Schirmer et al., 2001;
Beinker et al., 2002; Cashikar et al., 2002; Mogk et al., 2003b;
Strub et al., 2003). However, details of the Hsp100/ClpB reaction
cycle are unknown. The ATPase activity of both NBDs, as well as
an intact coiled-coil domain, are essential for chaperone activity
and in vivo thermotolerance (Kim et al., 2000a; Schirmer et al.,
2001; Hattendorf and Lindquist, 2002b; Mogk et al., 2003b). A
majority of data indicate that deletion of the N-terminal domain
does not impair measured activities, and its function remains
unknown (Clarke and Eriksson, 2000; Beinker et al., 2002). The
C-terminal domain is required for activity, but because deletion
of this domain prevents hexamerization, which is required for
ATPase activity, its direct role is not revealed by such experi-
ments. Studies with model substrates and effectors of the
ATPase activity have suggested that the C-terminal domain is
a substrate binding site (Smith et al., 1999), but others conclude
this domain modulates ATPase activity only (Strub et al., 2003).
The N-terminal domain, NBD1, and the coiled-coil domain have
all also been proposed to be involved in substrate interactions
(Dougan et al., 2002; Liu et al., 2002; Weibezahn et al., 2003).
However, the only data on binding of a natural substrate (TrfA to
Escherichia coli ClpB) reveals an interaction only with the surface
of NBD1 near the axial pore (Schlieker et al., 2004). Lindquist and
coworkers (Cashikar et al., 2002; Hattendorf and Lindquist,
2002a) proposed that in yeast Hsp104, stimulation of NBD2
activity drives a conformational change in the coiled-coil domain,
which in turn stimulates ATPase activity in NBD1. Cross-linking
studies with ClpB further suggest that movement of the coiled-
coil domain is necessary for chaperone activity in vitro (Lee et al.,
2003). How these motions are connected to protein disaggre-
gation remains to be defined. It has been suggested that ClpB
has a crowbar action in protein disaggregation, perhaps medi-
ated by the coiled-coil domain (Vale, 2000; Maurizi and Xia, 2004;
Weibezahn et al., 2004). Alternatively, or in addition, substrate
unfolding may be coupled to threading through the axial channel
of the ClpB hexamer in a mechanism related to that used by ClpA
(Ishikawa et al., 2001) and ClpX (Kim et al., 2000b) for protein
unfolding and transfer to the ClpP protease. Recent experiments
indicate mutations that affect protein loops in the axial channel
eliminate yeast Hsp104 function in protein disaggregation,
perhaps because of disruption of protein threading through the
channel (Lum et al., 2004).
We have taken a genetic approach to investigating the function
and mechanism of Hsp100/ClpB in Arabidopsis. In previous
work we showed that AtHsp101, encoded by the HOT1 gene, is
a major component controlling acquired thermotolerance, based
on the phenotypes of missense and protein null alleles, hot1-1
(E637K; in NBD2) and hot1-3 (a T-DNA insertion in exon 1),
respectively (Hong and Vierling, 2000, 2001). Loss of AtHsp101
function, however, does not appear to have major effects on
plant growth and development under optimal temperature
Figure 1. Location and Phenotype of Mutations in AtHsp101 (See Also
Supplemental Figure 1 Online).
(A) Location of hot1 mutations with a thermotolerance phenotype on
a schematic diagram of the AtHsp101 protein. The conserved AAA
modules consist of two domains, a nucleotide binding domain (NBD1 or
2), containing conserved motifs [Walker A, Walker B, Sensor 1(S1), and
Arg finger (R)], and a C-terminal small domain (gray boxes), containing
Sensor 2 (S2) in AAA2. The coiled-coil domain contains two signature
motifs (Lee et al., 2003), with hot1-4 located in signature motif II.
(B) Location of missense mutations that are wild type for thermotoler-
ance, which were obtained by Tilling analysis (see Methods).
(C) Quantitative assessment of thermotolerance in hot1 mutant seedlings
compared with their corresponding wild type (Columbia for hot1-1 and
hot1-4, Columbia erecta for all others). After growth for 2.5 d in the dark
at 228C, seedlings were pretreated at 388C for 90 min and returned to
228C for 2.5 d (388C samples), or pretreatment was followed by 2 h at
228C then 2 or 3 h at 458C before 2.5 d of recovery (388C>458C samples).
hot1-4 seedlings are more sensitive to 388C treatment than wild-type or
null alleles. Asterisk s indicate values equal to zero. Mean and standard
deviations were derived by measurement from three independent ex-
periments performed with 60 or more seedlings per mutant and values
plotted as a percentage of the 228C value.
(D) Protein gel blot analysis of AtHsp101 protein levels in wild-type and
hot1 seedlings after treatment at 388C.
560 The Plant Cell
conditions. To dissect further the mechanism of Hsp101 action in
thermotolerance, we sought additional mutant alleles either by
direct screening for mutants in a seedling thermotolerance assay
(Hong and Vierling, 2000) or by analysis of point mutations in
AtHsp101 obtained through the Arabidopsis Tilling Resource
(http://tilling.fhcrc.org:9366). We describe four new mutant al-
leles of Hsp101 that provide insight into structure and function of
this class of chaperone ATPase. One mutant allele, hot1-4, which
is a missense mutation in the coiled-coil domain (A499T) pro-
vides direct in vivo evidence for an essential function of this
unique domain. Using this allele, we then screened for suppres-
sor mutations and defined 13 intragenic suppressors. The
location of these suppressor mutations defines features essen-
tial for in vivo activity of Hsp101 and provides in vivo evidence for
a novel mechanistic link between the coiled-coil domain and the
axial channel of Hsp100/ClpB proteins. Furthermore, certain
suppressors restore the solubility of small heat shock proteins
(sHsps) after heat stress in the hot1-4 mutant, providing genetic
evidence for interaction of the Hsp100/ClpB and sHsp chaper-
one systems in higher plants.
RESULTS
AtHsp101 Mutants with Defects in Thermotoleran ce
Arabidopsis seedlings will continue to grow after a 458C, 2-h heat
treatment if first preconditioned at 388C for 90 min followed by 1
to 2 h at 228C. This ability to acclimate to 458C is lost in the
previously characterized missense (hot1-1; E637K) and protein
null alleles of AtHsp101 (hot1-3; T-DNA insertion in exon 1) (Hong
and Vierling, 2000, 2001). We used this assay to screen for
additional AtHsp101 mutants in a population of mutagenized
Arabidopsis seedlings and also to assay identified point muta-
tions in AtHsp101 from the Arabidopsis Tilling Resource (http://
tilling.fhcrc.org:9366) for loss of thermotolerance. From our
screen for additional thermotolerance mutants (Hong et al.,
2003), only one other mutant was recovered that failed to
complement hot1-1 in allelism tests and that mapped to the
Hsp101 chromosomal region (data not shown). Sequencing of
the AtHsp101 gene from this mutant identified a single missense
mutation resulting in an exchange of Thr for Ala-499 in the coiled-
coil domain (Figure 1A; see Supplemental Figure 1 online).
Analysis of the AtHsp101 tilling mutants uncovered three addi-
tional hot1 alleles that were defective in thermotolerance, R706K
(hot1-5), E509K (hot1-6), and R815D (hot1-7), all of which lie in
AAA module 2 (Figure 1A). The locations of an additional 35
missense mutations obtained by tilling showed no defect in
thermotolerance and are indicated in Figure 1B.
The relative severity of each of the newly identified defective
alleles (hot1-4 to hot1-7) was compared with the wild type and
the functionally null hot1-1 allele (Hong and Vierling, 2001) in
a quantitative assay for seedling thermotolerance (Figure 1C).
The hot1-4 and hot1-5 alleles are the most severe, showing no
ability to acclimate to treatment at 458C for 2 h, whereas the hot1-6
and hot1-7 alleles have a less severe phenotype, with hot1-6
showing the mildest phenotype. Differences in severity are not
because of differences in protein levels as all the alleles accu-
mulate AtHsp101 protein to wild-type levels (Figure 1D). There-
fore, we conclude that relative thermotolerance of the alleles
reflects the degree to which each mutation disrupts AtHsp101
function in vivo and defines critical AtHsp101 residues in the
coiled-coil domain and NBD2.
hot1-4 Is a Semidominant, Gain-of-Function
AtHsp101 Allele
We noted that in contrast with all the other hot1 alleles, hot1-4
was not only defective in acquired thermotolerance to 458C
treatment, but also showed sensitivity to the 388C acclimation
pretreatment (Figure 1C), which is a permissive treatment for the
other hot1 alleles. To determine the extent of this sensitivity,
elongation of hot1-4 seedlings was compared with the wild type,
protein null (hot1-3), and the missense hot1-1 alleles after in-
creasing time at 388C. The hot1-4 mutant displays dramatic
sensitivity to 388C treatment compared with the wild type and the
other loss-of-function alleles (Figure 2). The fact that the pheno-
type of hot1-4 at 388C is worse than the absence of the protein
(hot1-3 phenotype, Figure 1D) defines hot1-4 as a gain-of-
function mutation and indicates that the hot1-4 protein interferes
with essential cell functions at 38 8C. Furthermore, whereas the
hot1-5, hot1-6, and hot1-7 as well as the previously isolated
AtHsp101 alleles are recessive, loss-of function mutants, the
hot1-4 phenotype proved to be semidominant (Table 1). Com-
pared with wild-type and hot1-4 homozygous seedlings, hetero-
zygous hot1-4 plants showed an intermediate hypocotyl length
after a 458C heat treatment with preconditioning. Despite the
sensitivity of hot1-4 to mild heat treatment, the hot1-4 mutation
had no obvious effect on growth and development in the
absence of stress (data not shown).
To confirm that the hot1-4 mutant protein causes the observed
heat-sensitivity phenotypes, we cloned a hot1-4 mutant genomic
fragment, including its own 1.5-kb promoter region, and trans-
formed wild-type Arabidopsis plants with the mutated gene. As
Figure 2. Unusual Sensitivity of hot1-4 to 388C.
Hypocotyl elongation of hot1 mutant seedlings treated for different times
at 388C, as measured after an additional 2.5 d of growth in the dark at
228C. Replicated measurements performed as in Figure 1C.
AtHsp101 Structure and Thermotolerance 561
shown in Figure 3, the phenotype of hot1-4 can be recapitulated
by expression of the hot1-4 mutant protein in wild-type plants.
Protein gel blot analysis showed that levels of HSP accumulation
were unaffected in the transgenic plants. Thus, the hot1-4 mutant
protein functions in a dominant-negative fashion. In total, these
results provide evidence that the coiled-coil domain is required
for AtHsp101 function during an early step in the development of
thermotolerance. In addition, the semidominant, gain-of-function
phenotype of hot1-4 suggests that this mutation results in a
nonproductive interaction with a limiting substrate or cofactor.
Isolation of Suppressors of ho t1-4
The dominant-negative, 388C-sensitive phenotype of hot1-4
provided an excellent opportunity to screen for suppressor
mutations that could restore growth. Suppressor mutations
should provide additional insight into structure-function relation-
ships of AtHsp101 and potentially identify important AtHsp101
substrates or cofactors. Approximately 7500 homozygous
hot1-4 seeds were mutagenized with ethyl methanesulfonate,
and nearly 110,000 dark-grown, 2.5-d-old seedlings from the
M2 generation were screened for continued growth after a 2-h
treatment at 388C. In total, 43 lines were isolated that showed
a significant reversion of hot1-4 sensitivity to 388C treatment and
that exhibited the same phenotype in the subsequent M3
generation (data not shown).
To separate intragenic from extragenic suppressors, we
sequenced the AtHsp101 gene from all 43 suppressors. Seven
of the suppressors did not carry a second mutation in the
AtHsp101 gene and are unlikely to be linked to the original
hot1-4 mutation. Presumably, they represent extragenic sup-
pressors of hot1-4. These suppressors could define factors
specific to plant stress tolerance and/or identify genes that
encode AtHsp101 cofactors or critical substrates. Further ge-
netic and molecular analysis of these mutants is in progress. The
other 34 suppressors had a second mutation in the hot1-4 gene,
resulting in 13 different amino acid substitutions: eight in the first
nucleotide binding domain (NBD1) (R223K, A270T/A305T,
A297T, G313D, E319K, A329V, G384S, and P388S), four within
NBD2 (G611D, G649E, G653E, and A723V), and one in the
C-terminal region (V813M) (Figure 4).
To confirm that the loss of sensitivity to a 388C treatment in
mutants carrying a second site mutation in the Hsp101 gene was
because of this mutation and not to another extragenic mutation,
a plant carrying each of the individual mutations was crossed
to either the wild type or hot1-4. Segregation of the wild type,
hot1-4, and suppressed phenotype was then scored in the F1
and F2 generations for each cross. As shown in Table 2 and Sup-
plemental Tables 1 and 2 online, suppression of the 388C pheno-
type was recessive and linked to the hot1-4 mutation. These data
confirm that the second site mutation in Hsp101 is the cause of
the recovery of wild-type growth at 388C for each of these
suppressors.
The Suppresso r Mutations Include Both Loss-of-Fun ction
and Restoration-of-Function Alleles
The phenotypes of intragenic suppressor mutants, representing
each amino acid substitution, were retested at 388C to quantify
the degree of phenotypic reversion. All the intragenic suppressors
Table 1. Genetic Analysis of hot1 Mutants
Cross Generation Total WT
Semi-
dominant hot x
2
P
hot1-1 F1 23 23 0 0
3 WT F2
a
159 127 0 32 1.58 >0.1
hot1-3 F1 31 31 0 0
3 WT F2 252 187 0 65 0.08 >0.5
hot1-4 F1 30 0 30 0
3 WT F2 268 64 134 70 0.26 >0.5
hot1-5 F1 7 7 0 0
3 WT F2 42 33 0 9 0.28 >0.5
hot1-6 F1 8 8 0 0
3 WT F2 94 72 0 22 0.12 >0.5
hot1-7 F1 4 4 0 0
3 WT F2 48 38 0 10 0.44 >0.5
The phenotypic scoring was based on the hypocotyl elongation phe-
notypes of the parental lines after 458C for 2 h with pretreatment (388C
for 90 min). WT, wild type.
a
The hypocotyl length ranges of the phenotypic classes of F2 progenies
were 6.5 to 8 mm for the wild-type phenotype, 2.0 to 4.0 mm for the
semidominant phenotype, and 0 mm for hot1-4 mutant phenotype after
heat stress.
Figure 3. Expression of Mutant HOT1-4 Protein in Wild-Type Plants
Recapitulates the 388C Sensitivity Phenotype of hot1-4.
The hot1-4 genomic DNA under its own promoter was expressed in
transgenic wild-type plants. Two independent transgenic lines (T3:1 and
T3:2) are shown compared with the wild type and hot1-4 (three seedlings
each). The hypocotyl elongation assay was performed as in Figure 1C.
Equal protein samples from heat-stressed seedlings (388C for 90 min
followed by 2 h at 228C) were analyzed with the indicated antisera against
AtHsp101, sHsps, or glyceraldehyde-3-phosphate dehydrogenase
(GAPC).
562 The Plant Cell
showed a strong restoring phenotype of hypocotyl elongation
after the same 388C treatment that was used for their initial
identification (Table 3). In addition, when tested for sensitivity to
388C as 10-d-old seedlings, the suppressors also showed re-
covery of the wild-type phenotype (Figure 5A). Thus, all of the
suppressors clearly eliminated the dominant-negative function
of the hot1-4 allele at two distinct growth stages.
The intragenic suppressor mutants were then tested to de-
termine if the function of AtHsp101 in acquired thermotolerance
had been restored by measurement of hypocotyl elongation after
2hat458C of seedlings that had been preconditioned at 388C
(Table 3). The suppressors could clearly be separated into two
classes based on their response to this assay. Suppressors in
Class 1, which includes A270T/A305T, E319K, G384S, P388S,
G611D, A723V, and V813M, were completely arrested in hypo-
cotyl elongation after 2 h at 458C with preconditioning. Genetic
analysis confirmed that this phenotype also segregated with the
hot1-4 mutation, again confirming that it arises as a result of the
second site mutation in AtHsp101 (see Supplemental Tables 1
and 2 online). This acquired thermotolerance test indicates that
the Class 1 suppressors are most likely loss-of-function alleles
because they eliminate the dominant-negative phenotype of
hot1-4 but do not restore function in acquired thermotolerance.
By contrast, the second class of suppressors (Class 2) (R223K,
A297T, G313D, A329V, G649E, and G653E) partially restored
acquired thermotolerance of hypocotyl elongation, with the best
suppression shown by A329V, which elongated to
;75% of
wild-type levels (Table 3).
The same phenotypic behavior was observed when 10-d-old
suppressor seedlings were tested for acquired thermotolerance.
Class 1 suppressors failed to acquire thermotolerance; a 458C
treatment for 2 h (with preconditioning) completely blocked
production of additional leaves, and existing leaves and cotyle-
dons turned white (Figure 5B). Of the Class 2 suppressors, those
which restored hypocotyl elongation after 458C stress to <35% of
the wild type (G313D, G649E, and G653E; Table 3) did not exhibit
acquired thermotolerance at the 10-d-old seedling stage (Figure
5B). However, suppressors R223K, A297T, and A329V, which
showed stronger reversion of the hypocotyl phenotype (>43% of
the wild type), also showed acquired thermotolerance at the
Figure 4. Location of hot1-4 Suppressor Mutations in AtHsp101.
Class 1 loss-of-function suppressors are labeled in smaller gray font and
indicated with closed circles. Restoration-of-function Class 2 suppres-
sors are indicated in larger black font with open circles. The strongest
Class 2 suppressors, as discussed in the text, are underlined (see also
Supplemental Figure 1 online).
Table 2. Genetic Segregation Analysis of Intragenic Suppressors
Genotypes
No. of plants
x
2
P
Total WT hot Sup.
R223K F1 5 5 0 0
3 WT F2 36 36 0 0
R223K F1 5 0 0 5
3 hot1-4 F2 36 0 10 26 0.14 >0.9
A270/A305 F1 3 3 0 0
3 WT F2 38 38 0 0
A270/A305 F1 7 0 0 7
3 hot1-4 F2 40 0 9 31 0.13 >0.9
A297T F1 7 7 0 0
3 WT F2 36 36 0 0
A297T F1 6 0 0 6
3 hot1-4 F2 29 0 6 23 0.73 >0.5
G313D F1 5 5 0 0
3 WT F2 36 36 0 0
G313D F1 6 0 0 6
3 hot1-4 F2 32 0 7 25 0.16 >0.9
E319K F1 7 7 0 0
3 WT F2 32 32 0 0
E319K F1 2 0 0 2
3 hot1-4 F2 28 0 5 23 0.76 >0.5
A329V F1 5 5 0 0
3 WT F2 46 46 0 0
A329V F1 5 0 0 5
3 hot1-4 F2 39 0 8 31 0.14 >0.9
G384S F1 6 6 0 0
3 WT F2 36 36 0 0
G384S F1 5 0 0 5
3 hot1-4 F2 32 0 7 25 0.16 >0.9
P388S F1 10 10 0 0
3 WT F2 40 40 0 0
P388S F1 3 0 0 3
3 hot1-4 F2 40 0 9 31 0.13 >0.9
G611D F1 4 4 0 0
3 WT F2 46 46 0 0
G611D F1 5 0 0 5
3 hot1-4 F2 43 0 10 33 0.07 >0.9
G649E F1 5 5 0 0
3 WT F2 45 45 0 0
G649E F1 6 0 0 6
3 hot1-4 F2 44 0 8 36 0.36 >0.5
G653E F1 5 5 0 0
3 WT F2 37 37 0 0
G653E F1 5 0 0 5
3 hot1-4 F2 34 0 7 27 0.35 >0.5
A723V F1 5 5 0 0
3 WT F2 27 27 0 0
A723V F1 8 0 0 8
3 hot1-4 F2 35 0 8 27 0.08 >0.9
V813M F1 5 5 0 0
3 WT F2 46 46 0 0
V813M F1 5 0 0 5
3 hot1-4 F2 48 0 9 39 1 >0.5
Phenotype scoring was done by comparing the hypocotyl length
distribution patterns of the F2 seedlings with those of the parental lines.
The hypocotyl length ranges of the phenotypic classes of F2 progenies
were 9.5 to 10.5 mm for the wild-type phenotype, 9.0 to 11.0 mm for the
suppressor phenotype (Sup.), and 2.0 to 4.0 mm for hot1-4 mutant
phenotype after 388C heat treatment for 2 h. WT, wild type.
AtHsp101 Structure and Thermotolerance 563
10-d-old seedling stage (Figure 5B). Furthermore, the A329V
suppressor recovered as much as the wild type, whereas the
R223K and A297T suppressors had pale green leaves and were
smaller than the A329V suppressor, also consistent with the
relative growth of these suppressors in the hypocotyl assay.
Thus, the strong suppressors, R223K, A297T, and A329V, clearly
compensate for the primary functional defect caused by hot1-4,
although with somewhat different effectiveness. These three
mutations are all located in NBD1 (Figure 4), supporting a func-
tional interaction of this domain with the coiled-coil domain
containing the primary hot1-4 mutation.
The V813M and G384S Suppressors Accumulate
Truncated AtHsp101
To determine whether the restored growth phenotype of the
suppressors was because of a dose effect of the mutant protein,
protein gel blot analysis was performed on protein samples from
heat-treated, 2.5-d-old dark-grown seedlings. Except for V813M
and G384S, all the mutant proteins accumulated to similar levels
when compared with the wild type and hot1-4 (Figure 6). Although
the Class 1 mutants V813M and G384S are obviously missense
mutations,
;90- and 41-kD bands, respectively, were detected
with AtHsp101 antibody in these samples (Figure 6). Because the
antibody is directed specifically to the N-terminal domain of
AtHsp101 (Hong and Vierling, 2001), these bands most likely
represent C-terminally truncated proteolytic fragments gener-
ated in vivo as a result of protease sensitivity near the mutation
site. Thus, these two suppressor mutations clearly produce
a nonfunctional protein. This result supports the above conclu-
sion that the defective phenotype in acquired thermotolerance of
the Class 1 suppressors is caused by loss of AtHsp101 function.
Strong Suppressors Resto re the Function of AtHsp101 in
Protein Resol ubiliz atio n
The current model for Hsp100/ClpB function in thermotolerance
proposes that these proteins are required for reactivation of
aggregated proteins after heat stress (Glover and Lindquist,
1998; Goloubinoff et al., 1999; Motohashi et al., 1999). Therefore,
it can be assumed that during recovery from heat stress, heat-
aggregated proteins requiring AtHsp101 function should remain
in the insoluble cell fraction in hot1 mutants, whereas these
proteins should return to the soluble fraction in the wild type. It
also follows that if resolubilization is an essential function of
AtHsp101 for acquired thermotolerance, the strong hot1-4
suppressor mutations should restore protein solubility after
heat stress. As a first step to test this hypothesis, we sought
conditions for comparing the insoluble protein fractions from
wild-type and mutant plants during recovery from heat stress,
along with a suitable protein to assay for solubility. We de-
termined that in wild-type plants, the cytosolic Class I and II
Figure 5. Thermotolerance Phenotype of 10-d-Old Suppressor Mutants.
After growth for 10 d in the light at 228C, seedlings were treated either at
388Cfor3h(A) or at 388C for 90 min followed by 2 h at 228C and then 2 h
at 458C (B). Seedlings were photographed 7 d after treatment.
Table 3. Hypocotyl Elongation Phenotype of Intragenic Suppressors
Genotype
No. of
Isolates
Hypocotyl Length (%)
a
388C388C/458C(%)
WT 91.8 6 9.6 68.0 6 9.2 (100)
hot1-4 (A499T) 26.2 6 4.5 0 6 0
hot1-3 (null) 90.8 6 9.6 0 6 0
Class 1 suppressors
A270T/A305T 6 89.9 6 6.4 0 6 0
E319K 1 84.7 6 6.6 0 6 0
G384S 3 89.7 6 8.3 0 6 0
P388S 3 87.5 6 6.8 0 6 0
G611D 5 90.1 6 6.1 0 6 0
A723V 3 79.4 6 9.8 0 6 0
V813M 2 91.4 6 8.3 0 6 0
Class 2 suppressors
R223K 2 83.6 6 5.1 28.7 6 5.8 (42)
A297T 2 82.1 6 9.8 29.3 6 6.3 (43)
G313D 3 75.3 6 6.7 9.7 6 4.1 (14)
A329V 1 94.1 6 6.2 51.3 6 6.7 (75)
G649E 1 86.7 6 8.0 20.9 6 6.8 (30)
G653E 2 85.2 6 7.3 24.1 6 7.3 (35)
a
Dark-grown, 2.5-d-old seedlings were total hypocotyl elongation after
the heat stress was measured as a percentage of the 228C value. Mean
and standard deviations were derived by measurement from three
independent experiments performed with 60 or more seedlings per F3
seedling. The numbers in parentheses indicate percentage of elongation
compared with that of wild-type plants.
564 The Plant Cell
sHsps, partitioned during 458C heat stress into the insoluble cell
fraction and almost completely transitioned back to the soluble
fraction after 3 h of recovery from the heat stress (data not
shown). We therefore compared the solubility of either Class I
AtHsp17.6C-I or Class II AtHsp17.6C-II in wild-type, hot1-4, and
the strong suppressors, R223K, A297T, and A329V, after 3 h of
recovery from a 388C, 3-h heat stress or from a 458C, 45-min heat
stress with preconditioning. In contrast with the wild type,
a majority of the sHsps remained in the pellet fraction during
recovery from 458C stress in the hot1-4, hot1-1, and hot1-3
mutants (Figure 7). The mutants also showed detectably more
pelleted sHsps after the 388C stress. We then examined the
solubility of these sHsps in the strong suppressors. sHsp solu-
bility was directly correlated with the strength of the suppressor
(Figure 7), with the most effective suppressor, A329V, being
almost identical to the wild type and the weaker suppressors,
R223K and A297T, showing slightly more sHsp retained in the
insoluble fraction. The same samples were also used to examine
the solubility of AtHsp101. In contrast with the sHsps, neither the
wild type nor the hot1-1 allele of AtHsp101 becomes insoluble
during heat stress; similar behavior is reported for E. coli ClpB
(Mogk et al., 2003a, 2003c). A fraction of hot1-4 becomes
insoluble during heat stress, but insolubility does not correspond
to the phenotype, as at 388C the hot1-4 phenotype is already
severe, whereas little protein is insoluble. Only the strongest
suppressor reverses the AtHsp101 insolubility. Altogether, these
results support the model that one essential thermotolerance
function of AtHsp101 involves protein disaggregation and ge-
netically link Hsp101 to the sHsp chaperone system.
DISCUSSION
We have been able to identify features critical to Hsp100/ClpB
function in vivo using a sensitive, quantitative bioassay for
thermotolerance. We identified four missense mutations in the
Hsp100/ClpB protein, Arabidopsis AtHsp101, that compromise
the function of AtHsp101 in thermotolerance, including an un-
usual allele, hot1-4 (A499T), which shows a semidominant, gain-
of-function sensitivity to a mild, usually nonlethal heat stress.
This mutation, which lies in the coiled-coil domain of AtHsp101,
supports an essential role for this structural domain in Hsp100/
ClpB action in vivo. We also suggest that the gain-of-function
phenotype of hot1-4 is consistent with the interpretation that the
mutation disrupts AtHsp101 activity such that a critical substrate
or cofactor becomes limiting for the cell. Locations of the primary
missense mutations hot1-5, -6, and -7 and of suppressors of
hot1-4 define other features essential for in vivo function of
AtHsp101 and point to protein domain interactions required for
Hsp100/ClpB action. Finally, appearance of sHsps in the soluble
cell fraction during recovery from heat stress does not occur in
hot1-4 but is restored by strong suppressor mutants, in line with
the model that disruption of protein aggregates is an essential
AtHsp101 function in thermotolerance and supporting the model
that sHsps and Hsp100/ClpB proteins interact in a chaperone
network in the cell.
The coiled-coil domain of Hsp100/ClpB is unique to this class
of AAAþ proteins (Schirmer et al., 1996; Celerin et al., 1998).
Although referred to alternatively as the linker, middle region, or
spacer between AAA modules 1 and 2, it is actually an insertion
into the C-terminal small domain of AAA module 1 (Figures 1 and
8; see Supplemental Figure 1 online). Deletion of the coiled-coil
domain from E. coli ClpB has shown that it is not essential for
hexamerization or ATPase activity but is required for thermotol-
erance in vivo and for disaggregation of firefly luciferase in vitro
(Mogk et al., 2003b). The structure of T. thermophilus ClpB
reveals that this domain is formed by four helices (L1 to L4) that
Figure 6. Accumulation of AtHsp101 Protein in the Suppressor Mutants.
Protein samples from heat-stressed seedlings (388C for 90 min followed
by 2 h at 228C) were analyzed with AtHsp101 antiserum.
(A) Class 1 suppressor mutants.
(B) Class 2 suppressor mutants.
Figure 7. Reversal of sHsp Insolubility in the Suppressor Mutants.
Ten-day-old seedlings were treated either at 388C for 3 h (38) or at 388C
for 90 min followed by 2 h at 228C and then 60 min at 458C (45), and total
protein (T) was isolated after further recovery for 3 h at 228C. The
insoluble protein fraction (P) was separated from the soluble fraction (S)
by centrifugation and analyzed with AtHsp101, AtHsp17.6C-I (sHsp I),
and AtHsp17.6C-II (sHsp II) antiserum.
AtHsp101 Structure and Thermotolerance 565
interact in two coiled-coil motifs comprised of a-helices L1 and
L2, and L2 with L3/4 (Figures 8 and 9). Both coiled-coils have
features typical of Leu zippers. The hot1-4 mutation (A499T) lies
in helix L3, within the conserved sequence segment 494YDLAR-
AADL502 (484YDLNRAAEL492, in T. thermophilus) (Figures 8A
and 8B). This sequence motif was previously identified by
Schirmer et al. (1996) as characteristic of Hsp100/ClpB chaper-
ones. The weak mutation hot1-6 (E509K) lies in the coiled-coil
domain directly outside this conserved motif (Figure 8B). Neither
the hot1-4 nor hot1-6 residues are predicted to contact residues
in neighboring subunits, which along with the normal accumula-
tion of the proteins and the similar solubility of hot1-4 to the wild
type, suggest that these mutations do not significantly disrupt the
hexamer structure. In vitro cross-linking experiments also pro-
duce the same large, presumably oligomer forms of AtHsp101 in
the wild type and the hot1-4 mutants (U. Lee, unpublished data).
Eight other mutations in this domain have no effect on AtHsp101
function (Figure 1). Altogether, the hot1-4 and hot1-6 mutations
provide direct genetic evidence for the importance of helix L3 and
the coiled-coil domain in AtHsp101 function.
Interestingly, in a screen for mutants in Saccharomyces
cerevisiae Hsp104, Schirmer et al. (2004) recently reported
isolation of a mutation in the same motif, at the adjacent Ala
residue (A503V; 497YDTATA
ADL505 in S. cerevisiae). The
Hsp104 A503V mutant was an active ATPase in vitro (Cashikar
et al., 2002), exhibiting actually an approximately twofold
Figure 8. Location of the hot1 Mutations on the T. thermophilus ClpB Structure.
(A) The available structure of the T. thermophilus ClpB monomer (PDB file 1QVR; chain in [A]) is shown with the coiled-coil domain and AAA modules
indicated. Within each AAA module, the NBD domain is colored blue, with the Walker A and B motifs in orange, sensor 1 in pink, and the Arg finger in
yellow (see also Supplemental Figure 1 online). The small, a-helical domain of each AAA module is shown in gray. Nucleotide (AMP-PNP) is space-filled
in CPK coloring (hydrogen, white; oxygen, red; phosphorous, orange; sodium, blue). Motif 1 as defined by Schirmer et al. (1996) is colored purple in the
coiled-coil domain. The residue corresponding to each of the hot1 mutation sites is space-filled and colored green.
(B) Alternative view of the motif II region of the coiled-coil domain indicating the L2, L3, and L4 helices and the residue positions of the hot1-4 and hot1-6
mutations (in CPK coloring).
(C) View of interactions of the hot1-5 position (in CPK coloring) with residues within NDB2 and NBD1 as discussed in the text.
(D) Position of hot1-7 (CPK coloring) relative to the NBD2 ATP binding site. The sensor 2 Arg residue, which is part of the GAR motif, as well as sensor 1
are shown in pink. Residue numbering corresponds to AtHsp101. Figure was prepared with Swiss PDB viewer.
566 The Plant Cell
increase in basal ATPase activity (i.e., activity in the absence of
substrate). Therefore, the A503V mutation cannot disrupt the
hexameric structure, which is required for ATPase activity. This
observation further supports our interpretation that the hot1-4
mutant protein retains its hexameric structure. Furthermore, like
hot1-4, the Hsp104 A503V mutation also exhibits a gain-of-
function phenotype, leading to cell lethality when induced at the
normally permissive temperature of 378C (Schirmer et al., 2004).
The similarity of phenotype of these AtHsp101 and yeast Hsp104
mutations, along with the ability of AtHsp101 to support thermo-
tolerance in yeast (Schirmer et al., 1994), argue that these
proteins are true functional homologs.
The other mutations studied also provide new information
about interactions within the Hsp100/ClpB proteins. The strong
hot1-5 allele alters a residue (R706K) that is conserved in E. coli
(R705) and T. thermophilus (R695), and which is in a loop
between b-strands 4 and 5 in NBD2, positioned away from the
active site (Figures 8A and 8C; see Supplemental Figure 1 online).
Normal accumulation and solubility of the hot1-5 protein argues
that the protein is not significantly compromised structurally.
Based on the T. thermophilus structure, this Arg residue makes
intramolecular contact with three other residues conserved in
Arabidopsis and E. coli, including a Leu residue directly adjace nt
to sensor 1 in NBD1, and has side chain interactions with
conserved Glu and Arg residues in NBD2 helix D7 (Figure 8C).
Notably, a Lys residue is present at the hot1-5 position in yeast
Hsp104, and correspondingly the predicted interacting residues
are different. The unique importance of hot1-5 interactions, as
opposed to variation in the loop itself, is also indirectly supported
by the absence of a phenotype for another mutation in the loop
connecting b-strands 4 and 5, G703E (Figures 1 and 8C).
Hattendorf and Lindquist (2002a) have proposed that ATP hydro-
lysis at NBD1 depends on the nucleotide bound at NBD2 in yeast
Hsp104. We speculate that hot1-5 is in a position involved in
communicating nucleotide status between NBD2 and NBD1 and
note that the distance between the hot1-5 Arg residue and the
conserved Leu in NBD1 varies in the three ClpB molecules in the
crystal structure, consistent with a dynamic interaction.
Figure 9. Location of the hot1-4 Suppressor Mutations on the T. thermophilus ClpB Monomer Structure.
The major domains and motifs of HCP100/ClpB are colored as indicated and described in Figure 8. The sites of Class 1 suppressor mutations are
space-filled dark gray, and sites of the Class 2 suppressor mutations are space-filled in red. The three strongest Class 2 suppressors are indicated in
bold and are underlined. For A297, G653, and G649, which are in unresolved segments of the structure, dotted lines are used to indicate the relative
positions of those segments. The arrow indicates the general position of the axial channel of the hexameric form of the protein. Residue numbering
corresponds to AtHsp101. Figure was prepared with Swiss PDB viewer.
AtHsp101 Structure and Thermotolerance 567
It is difficult to define the possible effects of some of the other
AtHsp101 mutations. The hot1-7 mutation represents a dramatic
change (G815D) in a conserved motif (GAR) that includes the
sensor 2 Arg residue already recognized as important for
function (Figure 8D). The residues in the sensor 2 motif are in
contact with nucleotides, and the Arg residue in yeast Hsp104 is
proposed to contribute binding energy, but not discrimination
between ATP and ADP (Hattendorf and Lindquist, 2002a).
Mutations in sensor 2, or of all three residues of the GAR motif,
do not severely disrupt hexamerization or ATPase activity, but
prevent ability to dissociate protein aggregates (Hattendorf and
Lindquist, 2002b; Mogk et al., 2003b). In vivo, the moderate
phenotype of hot1-7 is consistent with a moderate defect in
thermotolerance observed for an R to M mutation of sensor 2 in
yeast (Hattendorf and Lindquist, 2002b). The absence of phe-
notype for 35 other missense mutations analyzed is not surpris-
ing, given that many lie in nonconserved residues or represent
conservative changes (Figure 1; see Supplemental Figure 1
online). Perhaps the most surprising of the aphenotypic muta-
tions is P671L, which represents a Pro residue conserved not
only in NBD2 of all ClpB proteins, but also found in ClpA.
The unusual sensitivity of the hot1-4 allele to 388C, which is
otherwise permissive for wild-type and AtHsp101 null plants, led
us to focus further on analysis of this mutation. We performed
a screen that identified both loss-of-function (Class 1) and
restoration-of-function (Class 2) intragenic suppressor muta-
tions in AtHsp101. Genetic dissection of Hsp100/ClpB function
by intragenic suppressor analysis has not been performed in any
organism, and we are also unaware of any previous studies using
intragenic suppressors for dissection of protein mechanism in
higher plants. We argue that the seven Class 1 mutations that
eliminated the 388Cheatsensitivityofhot1-4, but did not restore
the acquired thermotolerance function, represent loss-of-function
AtHsp101 mutants. Consistent with this interpretation, one of
these suppressors is in the Walker A motif of NBD 2 (G611D),
and three others are predicted to affect the nucleotide binding
pocket (E319K, P388S, and A723V) (Figure 9), which should
result in defective hexamerization or ATP hydrolysis as demon-
strated in vitro for other Hsp100/ClpB proteins (Kim et al., 2000a;
Watanabe et al., 2002; Mogk et al., 2003b). These findings
suggest that in hot1-4, AtHsp101 retains normal ATP hydrolysis
activity and hexamerization and indicates that these properties
are essential for the dominant-negative effect of this allele. Two
other Class 1 suppressors, G384S and V813M, are both located
between the second and third helices in the C-terminal sub-
domain of NBD1 and NBD2, respectively (Figure 9; see Sup-
plemental Figure 1 online). Both of these mutations lead to
accumulation of a truncated AtHsp101 protein with sizes that
predict that in vivo cleavage occurs in close proximity to the
mutation. Proteolytic removal of the C terminus in V813M
supports the requirement of this domain for function, which as
stated before, may involve not only hexamerization, but also
effector binding (Smith et al., 1999; Strub et al., 2003). Although
our screen was not performed to saturation, it is notable that no
loss-of-function suppressors were obtained in the N-terminal
domain, which has also not been associated with an essential
function in other organisms (Clarke and Eriksson, 2000; Beinker
et al., 2002; Mogk et al., 2003b).
Whereas the coiled-coil domain is clearly essential for Hsp100/
ClpB function, its role in the catalytic cycle is not known. The
position of hot1-4 (A499T) (and A503V in S. cerevisiae Hsp104)
suggests that this mutation could disrupt interactions of L3 with
L2 within a subunit. Lee et al. (2003) have proposed that motion of
the coiled-coil domain is required for Hsp100/ClpB chaperone
activity. They introduced Cys residues into T. thermophilus ClpB
to effect cross-linking of the L2/L3-4 coiled-coil (in which hot1-4
is located) to NBD1 and observed in vitro that ATP hydrolysis
continued, but the protein disaggregation activity of ClpB was
lost. The three hot1-4 suppressors that restore >43% of acquired
thermotolerance of hypocotyl growth and also restore acquired
thermotolerance of 10-d-old seedlings are all located in NBD1
(R223K, A297T, and A329V). The strongest of these, A329V, has
>70% wild-type activity in the hypocotyl assay and restores
solubility of sHsps during recovery from heat stress. Interest-
ingly, A329V lies in close proximity to the NBD1 Arg finger, which
is believed to interact with the nucleotide in an adjacent subunit
(Figure 9). Another weak suppressor, G313D, is also in NBD1
(Figure 9). Taken together, the location of the Class 2 restoration-
of-function suppressors of hot1-4 is consistent with the inter-
pretation that dynamic interaction of the coiled-coil domain with
NBD1 is indeed essential in vivo.
How Hsp100/ClpB proteins act to disaggregate proteins has
been proposed to involve a crowbar action of the coiled-coil
domain and/or threading of substrate through the axial channel
of the hexamer, analogous to the mechanism of other hexameric
ATPases (Lee et al., 2003; Lum et al., 2004; Schlieker et al., 2004).
Location of the Class 2 suppressors provides potential support
for the latter mechanism. The strong suppressor A297V is
located in a presumably flexible, disordered segment that was
not resolved in the TtClpB structure, but which is in the axial
channel region. A329V, another strong suppressor, is positioned
to potentially alter interactions with another unresolved axial
channel loop (TtClpB residues 234 to 246; corresponding to 243
to 255 in AtHsp101), which has recently been proposed to
contain residues that bind the E. coli ClpB substrate TrfA
(Schlieker et al., 2004). The only restoration-of-function sup-
pressors outside NBD1 are the two weak suppressors, G649E
and G653E in NBD 2, which also lie in an unresolved axial channel
loop (TtClpB residues 638 to 650; corresponding to AtHsp101
648 to 660) (Figure 9; see Supplemental Figure 1 online). This
loop contains the conserved motif GYVG found in other AAAþ
proteins (with the first G corresponding to G653 in AtHsp101).
Mutation of the Tyr residue in this loop impairs function of E. coli
ClpX (Siddiqui et al., 2004) and HslU (Wang et al., 2001b).
Structural studies also indicate that this loop gates the axial
channel of HslU during the ATPase cycle (Wang et al., 2001a).
Recent experiments with yeast Hsp104 also support an essential
role for this loop in the protein disaggregation reaction (Lum et al.,
2004). In total, we suggest that these intragenic suppressors
point to a model in which motions of the coiled-coil domain
modulate the ATPase activity of NBD1 and the position of axial
channel loops in AtHsp101, which may be involved in threading
of substrate through the axial channel.
Analysis of the solubility of plant cytosolic sHsps in the hot1-4
mutant and the strong suppressors also provides data to
link these two chaperone systems in a eukaryote. Genetic
568 The Plant Cell
interactions of sHsps and ClpB have been reported in both E. coli
and Synechocystis (Giese and Vierling, 2002; Mogk et al., 2003a)
and are supported by in vitro analysis of E. coli ClpB activity in
combination with E. coli, Synechocystis, and plant cytosolic
class I sHsps (Mogk et al., 2003c). The current model for sHsp
function proposes that sHsps bind and maintain denaturing
proteins in a form that is accessible to ATP-dependent refolding
chaperones. Furthermore, their presence in large protein aggre-
gates makes these aggregates better substrates for the action of
Hsp100/ClpB (Mogk et al., 2003c). However, although Hsp100/
ClpB disaggregates sHsp containing complexes, there is no
evidence for direct physical interaction of Hsp100/ClpB with
sHsps (Mogk et al., 2003a, 2003c), and we did not observe
association of AtHsp101 with sHsps in the insoluble protein
fraction of the cell. In total, the data here indicate that the plant
cytosolic Class II, as well as cytosolic Class I sHsps, which
diverged on the order of 400 million years ago (Waters and
Vierling, 1999), may both facilitate protein disaggregation by
Hsp100/ClpB in plants.
At which step the hot1-4 mutation affects catalysis will require
additional biochemical studies. It does not appear that the
dominant-negative phenotype of hot1-4 is attributable to un-
regulated ATPase activity of the mutant AtHsp101 protein,
leading to depletion of cellular ATP. Measurement of ATP levels
in the wild type, hot1-3, and hot1-4 showed no significant
differences in ATP levels between the genotypes during heat
stress or recovery (U. Lee and E. Vierling, unpublished data). One
possibility is that the mutation traps AtHsp101 in a nonfunctional
protein complex such that another critical cofactor or substrate
becomes limiting for the cell. Such a complex must still be
soluble, as our analysis does not indicate that a significant
fraction of AtHsp101 becomes insoluble at 388C where the
hot1-4 phenotype is still severe. The Class 1 suppressors that
eliminate the dominant-negative phenotype are predicted to
disrupt the basic hexameric structure and ATPase activity,
consistent with the requirement of these activities to form
a trapped complex. By altering structural interactions of the
coiled-coil domain with NBD1, the strong Class 2 suppressors
may eliminate this trapped intermediate. Weaker Class 2 sup-
pressors may affect the protein’s conformation, but not enough
to fully disrupt the formation of the nonfunctional complex
between hot1-4 and its bound protein(s). Another possibility is
that the suppressors directly or indirectly alter a binding site for
substrate or functional partner protein, such that a nonfunctional
complex no longer forms, but sufficient substrate or cofactor
interactions are retained for partial function in vivo. Attempts to
coimmunoprecipitate hot1-4 in complex with other proteins has
not been successful. Biochemical characterization of hot1-4
along with identity of extragenic suppressors should provide
further insight into the nature of the hot1-4 defect and the
mechanism of Hsp100/ClpB action.
METHODS
Plant Material and Growth Conditions
Arabidopsis thaliana (Columbia accession) plants were grown on plates in
the dark or under long-day conditions (16 h light/8 h dark) in a controlled-
temperature growth chamber (228C/188C). Thermotolerance tests of
2.5-d-old dark-grown or 10-d-old light-grown seedlings were performed
basically according to Hong and Vierling (2000).
Isolation and Genetic Analysis of hot1 Muta nt Alle les
The hot1-4 mutation was isolated as a thermotolerance-defective mutant
in a screen based on hypocotyl elongation of 2.5-d-old dark-grown
seedlings (Hong and Vierling, 2000). Allelism tests showed that hot1-4
was tightly linked to the previously described AtHsp101 loss-of-function
missense allele, hot1-1 (Hong and Vierling, 2000). For most of the
experiments described in this work, the third backcrossed line of hot1-4
to Columbia wild type was used. Tilling analysis (in the Columbia ecotype,
carrying the erecta mutation) was performed on three segments of the
AtHsp101gene, encompassing approximately amino acid residues 1 to
230, 355 to 595, and 660 to 911 (Arabidopsis Tilling Resource, http://
tilling.fhcrc.org:9366) (see Supplemental Figure 1 online). The hot1-5,
hot1-6, and hot1-7 mutants were recovered from 37 missense mutations
in AtHsp101 obtained from this analysis. Three stop codon mutations
(Q409, Q422, and Q704) were also obtained, confirmed to be defective in
thermotolerance, and not studied further. Each line was assayed for
thermotolerance defects in the homozygous or heterozygous state, and
homozygous lines were isolated for lines showing a phenotype (hot1-5,
hot1-6,andhot1-7 ). One homozygous M4 plant from each mutant line
was then backcrossed to Columbia erecta wild-type plants, and one
homozygous F3 line for each mutation was used for quantitative analysis.
Isolation and Genetic Analysis of hot1-4 Suppressor Mutations
Approximately 7500 homozygous seeds of hot1-4 were mutagenized
with ethyl methanesulfonate, and
;110,000 M2 seed were screened for
suppressor mutants as follows. M2 seeds were surface-sterilized and
plated on minimal medium containing 0.5% sucrose. Plates were in-
cubated at 48C for 3 d and then placed in a vertical position at 228Cfor
2.5 d in the dark. The dark-grown seedlings were treated at 388Cfor2h,
and then the plates were returned to the dark at 228C for 1.5 d. Seedlings
that showed increased hypocotyl elongation compared with hot1-4 were
rescued by growth under light for approximately 1 week before being
transplanted to soil. The selected seedlings were retested for tolerance to
388C in the next generation (M3) as described above.
To distinguish if the suppressor mutations were intragenic or extra-
genic, the entire AtHsp101 gene from the candidate suppressors was
amplified by PCR using specific primers. The amplified DNA products
were sequenced on both strands. Thirty-four lines were found to contain
the original hot1-4 mutation together with an additional missense muta-
tion within the AtHsp101 coding region. The same suppressor mutation
was found in more than two independent lines (R223K, A270/A305T,
A297T, G313D, G384S, P388S, G611D, G653E, A723D, and V813M),
whereas E319K, A329V, and G649E mutations, which were isolated only
once, were confirmed by sequencing from two individual lines (Table 3).
To test genetic linkage between hot1-4 and the suppressors, the
homozygous M3 or M4 intragenic suppressors with second site muta-
tions in AtHSP101 were backcrossed to hot1-4. The F1 plants all showed
the suppressing phenotype after 388C heat treatment for 2 h. The
subsequent F2 progenies completely segregated in a 3:1 ratio of the
suppressor mutant to hot1-4, indicating that the suppressor mutations
are tightly linked to the hot1-4 mutation (Table 2; see Supplemental
Tables 1 and 2 online).
To obtain F3 homozygous intragenic suppressor lines for phenotypic
analysis, the homozygous M3 or M4 intragenic suppressors were out-
crossed to wild-type plants. All F1 plants showed the wild-type pheno-
type, and F2 progenies segregated either the hot1-4 mutant phenotype
(Class 1 suppressor) or the suppressor phenotype (Class 2 suppressor) at
AtHsp101 Structure and Thermotolerance 569
a ratio of 1:3 to the wild-type phenotype when tested by heating at 458C
for 2 h with pretreatment (388C for 90 min). Scoring of the hypocotyl length
phenotype in F2 progenies was based on the hypocotyl length distribu-
tion of the parental lines.
Plant Transformation
A 4.7-kb XhoI/XbaI AtHsp101 genomic region containing the hot1-4
mutation, including 1.5 kb of the promoter region, was cloned into pBin19
and transformed into Columbia wild-type plants by the floral dipping
method (Clough and Bent, 1998). A total of 20 lines (T1 generation) were
selected on minimal plates with kanamycin (30 mg/mL). The number of
T-DNA insertion loci was determined in the T2 generation based on the
segregation ratio of both kanamycin resistance and the hot1-4 pheno-
type. Two independent T3 homozygous lines were used for phenotypic
studies.
Fractionation of Heat-Denatured Proteins
Ten-day-old seedlings were pretreated at 388C followed by 2 h at 228C,
and then heat shocked at 458C for 60 min (denaturatio n phase), followed
by 3 h of recovery at 228C (recovery phase). Total protein was extracted
before the denaturation phase and after the recovery phase in non-
denaturing buffer (25 mM Hepes, pH 7.5, 0.5% Triton X-100, 200 mM
NaCl, 0.5 mM EDTA, 5 mM e-amino-N-caproic acid, and 1.0 mM
benzamidine). Protein concentration was determined using a Coomassie
Brilliant Blue dye binding assay (Ghosh et al., 1988) with BSA as
a standard. Total protein solutions were immediately centrifuged at
19,000g for 15 min at 48C. Supernatants were mixed with the same
volume of SDS sample buffer (60 mM Tris-HCl, pH 7.5, 60 mM DTT, 2.0%
[w/v] SDS, 15% [w/v] sucrose, 5 mM e-amino-N-caproic acid, and 1.0 mM
benzamidine). The pellet was suspended in nondenaturing buffer, washed
six times, and then resuspended to the original volume in SDS sample
buffer. Proteins were separated by SDS-PAGE on 15% acrylamide gels
and processed for protein gel blot analysis (Hong and Vierling, 2001).
SDS-PAGE and Protein Gel B lot Analysis
The 2.5-d-old dark-grown seedlings were treated at 388C for 90 min, and
then total protein was extracted in SDS sample buffer and separated by
SDS-PAGE on 7.5% or 15% acrylamide gels and processed for protein
gel blot analysis (Hong and Vierling, 2001). Protein blots were probed with
rabbit antiserum against AtHsp101 or against AtHsp17.6C-I or -II (Hong
and Vierling, 2001). As a loading control, blots were probed for cytosolic
glyceraldehyde-3-phosphate dehydrogenase using a GAPC antibody
(gift of Ming-Che Shih, University of Iowa) as described (Chan et al., 2002).
Blots were incubated with goat anti-rabbit horseradish peroxidase and
bands visualized by enhanced chemiluminescence (Amersham Interna-
tional, Piscataway, NJ).
ACKNOWLEDGMENTS
We thank Ming-Che Shih for the glyceraldehyde-3-phosphate dehydro-
genase antibodies, K. Giese, M. Mishkind, and C. Dieckmann for
critique of the manuscript, and Joseph T. Carroll for preparation of the
structure figures. Supported by Department of Energy Grant DE-FG03-
99ER20338 to E.V. S.W.H. was in part supported by the Agricultural
Plant Stress Research Center (Kwang Ju, South Korea).
Received September 3, 2004; accepted November 11, 2004.
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