From the Cellular and Molecular Biology Program and
the Department of Horticulture, University of Wisconsin,
Madison, Wisconsin 53706 and the
Department of Environmental
Horticulture, Institute of Food and Agricultural Sciences, University
of Florida, Gainesville, Florida 32611-0670
Received for publication, September 20, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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Small
ubiquitin-like modifier (SUMO) is a member of
the superfamily of ubiquitin-like polypeptides that become covalently attached to various intracellular target proteins as a way to alter
their function, location, and/or half-life. Here we show that the SUMO
conjugation system operates in plants through a characterization of the
Arabidopsis SUMO pathway. An eight-gene family encoding the
SUMO tag was discovered as were genes encoding the various enzymes
required for SUMO processing, ligation, and release. A diverse array of
conjugates could be detected, some of which appear to be SUMO
isoform-specific. The levels of SUMO1 and -2 conjugates but not SUMO3
conjugates increased substantially following exposure of seedlings to
stress conditions, including heat shock, H2O2,
ethanol, and the amino acid analog canavanine. The heat-induced
accumulation could be detected within 2 min from the start of a
temperature upshift, suggesting that SUMO1/2 conjugation is one of the
early plant responses to heat stress. Overexpression of SUMO2 enhanced
both the steady state levels of SUMO2 conjugates under normal growth
conditions and the subsequent heat shock-induced accumulation. This
accumulation was dampened in an Arabidopsis line
engineered for increased thermotolerance by overexpressing the
cytosolic isoform of the HSP70 chaperonin. Taken together, the SUMO
conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in plants with initial
data indicating that one important function may be in stress protection
and/or repair.
Post-translational modifications of proteins play a critical role
in most cellular processes through their unique ability to alter
rapidly and reversibly the functions of preexisting proteins, multiprotein complexes, and intracellular structures. Although originally thought to be restricted to small molecules like phosphate and sugars, emerging data now show that several distinct types of
polypeptide tags are important modifiers as well (1-4). These polypeptides become covalently attached to various intracellular targets via mechanistically similar ATP-dependent reaction
cascades involving activation
(E1s)1 and conjugation (E2s)
enzymes. Sometimes an additional enzyme (E3s) also participates in
target recognition and ligation. Ultimately, a protein conjugate is
formed bearing the tag linked via an isopeptide bond between its
C-terminal glycine and free lysyl The most pervasive and best understood tag is the 76-amino acid
polypeptide ubiquitin (Ub) (5, 6). Its main function is to become
attached post-translationally to proteins destined for degradation. In
most cases, a poly-Ub chain is assembled on the target via reiterative
rounds of conjugation. These poly-ubiquitinated intermediates are then
recognized by the 26 S proteasome, a protease complex with broad
specificity that degrades the target with the concomitant release of
the Ub moieties undigested. In other cases, a single Ub is added. These
mono-ubiquitinated intermediates help affect vesicular trafficking
and shuttle short lived membrane proteins to the lysosome/vacuole where
they are degraded by resident proteases (7).
More recently, a family of polypeptides distantly related to Ub called
small ubiquitin-like modifiers
(SUMOs; also known as sentrin, Smt3, ULP, and PIC1) has emerged as a
second influential modifier (2-4). These ~100-amino acid tags are
only 8-15% identical to Ub but fold into a similar globular structure
with a flexible protruding C-terminal tail that ends in the glycine
necessary for target ligation (8). Like Ub, SUMOs are synthesized as inactive precursors that require processing by specific protease(s) to
expose this glycine. SUMOs then employ an E1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino groups within the target.
Depending on the tag and/or the target protein, the function, location,
and/or half-life of the target can be affected. A family of
tag-specific proteases also participates in each of the pathways. These
proteases help generate the active form of the tag by removing extra
residues that cap the C-terminal glycine of the polypeptide and/or are
used to disassemble conjugates by cleaving the isopeptide bond between
the tag and the target, thus releasing each in an unmodified form.
E2
E3 conjugation scheme similar to ubiquitination (Fig. 1)
but appear to impart unique non-proteolytic functions to the target
(2-4). Although most targets are modified by a single SUMO moiety,
others can bear chains of SUMOs polymerized by concatenation of the
individual SUMOs through a specific lysine in each unit (9).
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Fig. 1.
Description of the SUMO modification
pathway. The pathway begins with processing of the SUMO precursors
by ULPs to release the mature forms bearing an exposed C-terminal
glycine. The E1 heterodimer SAE1/SAE2 activates the SUMO in an
ATP-dependent reaction, resulting in binding of the SUMO
moiety through a high energy thiol ester linkage to a cysteine
(C) in the E1. The activated SUMO is then transferred to the
active-site cysteine in the E2 SCE1 by transesterification and finally
attached to the target protein with the help of E3s like SIZ1. The
conjugate is connected by an isopeptide bond between the C-terminal
glycine of SUMO and a lysine (K) in the target. ULPs can
also release the bound SUMO thus generating both proteins in free
unmodified forms.
By analysis of conjugation mutants and the identification of specific
targets, it has become apparent that SUMOs play an influential role in
various cellular processes in yeast and animals by regulating protein-protein interactions and subcellular location or by
antagonizing ubiquitination (2-4). For example, both SUMO and all the
enzymes in the ligation pathway are essential in yeast
(Saccharomyces cerevisiae), and the corresponding
temperature-sensitive mutants arrest at the G2/M
transition, implicating a critical role for SUMO in the cell cycle
(10-13). Sumolation of RanGAP1 in animals directs its relocation from
the cytosol to the nuclear pore complex where it activates the Ran
GTPase, a regulator of nuclear protein transport (14). A number of
DNA-binding proteins are SUMO targets, including p53, c-Jun, c-Myb,
AP-2, the androgen receptor, and the heat shock transcription factors
(HSF)-1 and -2 (15-21). In some cases sumolation localizes the
proteins to nuclear promyelocytic leukemia bodies or increases
their ability to activate gene expression. In mammalian culture cells,
the levels of SUMO conjugates are increased by heat shock, oxidative
stress, and DNA-damaging agents, suggesting a role in stress protection
(22, 23). Sequence comparisons of the various SUMO targets have
identified a consensus binding motif, KXE, where
is a
large hydrophobic amino acid, K is the lysine to which SUMO is
conjugated, X is any amino acid, and E is glutamic acid (2).
For the cytosolic inhibitor of NF-
B, I
B
, the same lysine can
be either sumolated or ubiquitinated, suggesting in some cases that
SUMO conjugation blocks Ub attachment and thus protects proteins from
degradation (24).
In plants, little is known about the SUMO pathway or the nature of its targets. In preliminary studies, we identified a family of Arabidopsis genes that encode proteins substantially similar to yeast and animal SUMOs (1). Immunoblots with anti-SUMO1 antibodies detected a plethora of SUMO conjugates in a variety of plant species, indicating that the conjugation cascade is present as well. More recent data suggested that SUMO conjugation plays an important role in plant defense. Hanania et al. (25) isolated a tomato SUMO gene in a yeast two-hybrid screen for proteins that interact with ethylene-inducing xylanase from the fungus Trichoderma viride. This xylanase is a strong elicitor of the rapid defense response in tomato. Orth et al. (26) identified AvrBsT in the plant pathogen Xantomonas campestris as a potential SUMO protease. AvrBsT enters its hosts via a type III secretion system where it then interferes with the host defense response, possibly by de-sumolating a key defense regulator.
To understand further the roles of sumolation in plants, we have begun
to characterize the SUMO pathway in Arabidopsis thaliana (1). Here we report the identification of many of the core components,
including eight genes encoding the full-length SUMO tag and the
predicted E1, E2, E3, and SUMO proteases required for SUMO processing,
ligation, and conjugate disassembly. Immunoblot analysis with
isoform-specific antibodies against SUMO1/2 and -3 detected different
profiles of conjugates in crude Arabidopsis extracts,
indicating that the individual SUMOs have distinct targets. The array
of SUMO1/2 conjugates are substantially increased in planta
by exposing seedlings to heat shock, H2O2,
ethanol, and canavanine, with the kinetics of accumulation suggesting
that sumolation by SUMO1/2 has an early role in the plant stress response.
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EXPERIMENTAL PROCEDURES |
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Identification and Analyses of SUMO Pathway Genes in Arabidopsis-- Arabidopsis genes encoding SUMOs (SUMs), SUMO-activating enzyme (E1: SAE1 and -2), SUMO-conjugating enzyme (E2: SCE), SUMO ligase (E3: SIZ1), and SUMO proteases (ULPs) were identified by Blast searches (27) of the A. thaliana DNA database (www.arabidopsis.org). Primary queries were human SUMO-1 (GenBankTM accession number X99585 (28)), SAE1 and SAE2 (GenBankTM accession numbers AF110956 and AF110957 (29)), S. cerevisiae UBC9 (GenBankTM accession number X82538 (11)), SIZ1 (GenBankTM accession number NP_010697 (30)), Smt4 (GenBankTM accession number AAA69556 (31)), and ULP1 (GenBankTM accession number Q02724 (32)). Gene structures for SUM1-3, SAE1a-b, SAE2, SCE1a-b, and SIZ1 were determined by comparison of chromosomal DNA sequences with those obtained from full-length cDNAs provided from the expressed sequence tag (EST) database or by reverse-transcribed (RT)-PCR. Sequences not yet described were deposited in the GenBankTM database under accession numbers AF510519 (SUM1), AF510520 (SUM2), AF510521 (SUM3), AF510522 (SUM5), AF510523 (SAE1a), AF510524 (SAE1b), and AF510525 (SAE2). The structures of the remaining loci were predicted from partial cDNAs (when available) and comparisons with Arabidopsis, yeast, and animal relatives.
Sequences were analyzed using the ClustalW algorithm (www2.ebi.ac.uk/clustalw). Multiple sequence alignments and neighbor joining analyses were displayed using SeqVu 1.0 (The Garvan Institute of Medical Research, Sydney, Australia) and the Njplot programs (mgouy{at}biomserv.univ-lyon1.fr), respectively. Protein domains were identified by the Smart prediction program (smart.embl-heidelberg.de), and sequence homology was detected by Block Maker (blocks.fhcrc.org/blocks/blockmkr/make_blocks.html).
Plant Growth Conditions and Stress Treatments-- A. thaliana ecotype Col-0 tissues were obtained from plants grown on soil at 22 °C in continuous light. For the stress treatments, plants were grown at 24 °C in liquid Gamborg's B5 medium (Invitrogen) in continuous light with shaking (100 rpm) after a 4 °C stratification for 2 days. To initiate temperature stress, cultures where transferred to shaking water baths preset to the designated temperatures. For chemical treatments, compounds were added directly into the liquid culture medium.
Immunological Analyses--
Antibodies were raised against
full-length recombinant Arabidopsis SUMO1 and -3. The
full-length SUM1 and SUM3 cDNAs were generated by PCR using either the cDNA (SUM1) or a
RT-PCR product from total RNA isolated from A. thaliana
Col-0 (SUM3) as the templates. The 5' and 3' primers were
designed to introduce NdeI and XhoI sites or
NdeI and SmaI sites at the predicted start and
stop codons, respectively. The SUM1 cDNA was inserted
into NdeI/XhoI-digested pET28b (Novagen, Madison,
WI) and mobilized into Escherichia coli strain BL21 (DE3).
Following a 3-h induction at 37 °C with 0.4 mM
isopropyl-1-thio--D-galactopyranoside, the clarified
extract was heated to 90 °C for 30 min and clarified, and the
supernatant was subjected to Sephadex G-75 column chromatography
(Amersham Biosciences). The SUMO1-containing eluate, which represented
a nearly homogeneous preparation as determined by SDS-PAGE, was injected directly into rabbits. For SUMO3, the RT-PCR product was
cloned into pGEM®-T Easy vector (Promega, Madison, WI), released as a
NdeI/SmaI fragment, and then introduced into the
pTYB2 vector linearized with SmaI and NdeI for
expression in E. coli as an intein/chitin-binding protein
fusion (IMPACT I, New England Biolabs). Following a 3-h induction at
37 °C, the BL21(DE3) cells were lysed, and total soluble protein was
applied to a chitin column according to manufacturer's recommendations
(New England Biolabs). The bound protein was treated with 30 mM dithioerythritol for 12 h at 4 °C. The intein
cleavage product was eluted, heated for 30 min at 90 °C, and
separated from precipitated proteins by centrifugation. The supernatant
was applied to a Sephadex G-75 column; the SUMO3-containing fractions were used directly as the antigen.
Anti-SUMO1 antibodies were affinity-purified from serum by adsorption to SUMO1 protein coupled to Affi-Gel 10 beads (Bio-Rad). Anti-SUMO3 antibodies were purified by protein A affinity chromatography (Sigma). The anti-Ub antibody, anti-PBA1 antiserum, and anti-HSP101 antiserum were described previously (33, 34).
Immunoblot analyses were performed as described (33), using proteins subjected to SDS-PAGE and transferred to nitrocellulose membranes (Hybond-C Extra, Amersham Biosciences). The membranes were blocked using 10% milk powder and probed with antibodies diluted in phosphate-buffered saline containing 1% milk powder. Detection employed either alkaline phosphatase- or peroxidase-labeled goat anti-rabbit immunoglobulins (Kirkegaard & Perry Laboratories, Gaithersburg, MD) in conjunction with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate or Super Signal chemiluminescence (Pierce) and X-Omat autoradiographic film (Eastman Kodak), respectively.
Immunolocalization was performed as described using thin sections of developing Arabidopsis embryos (35). Sections were probed with preimmune antiserum or anti-SUMO1 antibodies followed by fluorescein isothiocyanate-labeled goat antibodies against rabbit IgGs (Jackson ImmunoResearch Laboratories, West Grove, PA) and visualized by fluorescence confocal microscopy. DNA was detected by staining the sections with the TO-PRO 3 (Molecular Probes, Eugene, OR).
In Vitro Conjugation Reactions-- Purified recombinant SUMO1 was 32P-labeled using protein kinase A (Sigma) as described (36) and separated from unincorporated 32P by filtration on a NAP10 column (Amersham Biosciences). Plant extracts were prepared by homogenizing pulverized frozen tissue in 50 mM Tris-HCl (pH 8.0), 1 mM Na4EDTA, 300 mM sucrose, and 14 mM 2-mercaptoethanol (37). Reactions (100 µl) contained 0.5 µg of 32P-SUMO1 and either an ATP-regenerating or an ATP-depleting system (38). After a 1-h incubation at 25 °C, the reactions were quenched by addition of SDS sample buffer, and the products were visualized by autoradiography following SDS-PAGE.
Generation and Analysis of Plants Overexpressing SUMO2 and HSC70-- The coding region for SUM2 was PCR-amplified from the full-length cDNA using primers designed to introduce XbaI sites at both ends (underlined) 5'-CCCGCGAATCTCTCTAGAGGCTTCGCTTATATC and 3'-CCATTAAAATAATCTAGAGTAAAAGCAGAAGAGCTT. The product was digested with XbaI and inserted into the XbaI-linearized binary T-DNA vector pGSVE9 immediately downstream of the Cauliflower Mosaic Virus 35S promoter (33). The 35S-SUM2 construction was introduced into the Agrobacterium tumefaciens strain AtC58RifR(pMP90), which was then used to transform A. thaliana ectotype Col-0 by the floral dip method (33). Initial transformants and selfed T2 progeny were identified by hygromycin resistance. The T3 progenies homozygous for hygromycin resistance were confirmed to overexpress SUMO2 by immunoblot analysis with anti-SUMO1 antibodies.
Homozygous T3 and T4 plants overexpressing the HSC70-1
cDNA under the direction of the Figwort Mosaic Virus promoter and
the Nopaline Synthase 3' end were created (lines 8-7
and 8-9 as described).2 For
controls, wild-type Col-0 and a weak HSC70-OX transgenic (8-11) line
were used.2 The protein was detected with a monoclonal
antibody against spinach HSC70 (SPA-817, StressGen, Victoria, British
Columbia, Canada).
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RESULTS |
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A Family of Genes Encode Arabidopsis SUMO-- Previous studies by us and others (1, 25) indicated that plants express SUMOs and likely much of the associated conjugation/deconjugation pathway. For a more comprehensive analysis, we used yeast and animal genes encoding various SUMO pathway components as queries to search for orthologs in the Arabidopsis protein and DNA databases. Our initial search identified nine Arabidopsis SUM genes that encode all or part of the canonical SUMO sequence (Table I). The similarity of their intron/exon structures suggested that the Arabidopsis SUM family evolved recently from a common progenitor. With the exception of SUM5 and SUM9, each contains two introns interrupting the coding region at the same location (Fig. 2A). For SUM5, the first intron is absent. For SUM9, the first exon is absent; the remaining coding region contains a number of in-frame stop codons, suggesting that this locus is a pseudogene. Interestingly, six of the nine SUM loci (SUM2 and -3, SUM4 and -6 (on chromosome 5), and SUM7 and -8 (on chromosome 4)) are arranged as tandem pairs in the genome without apparent intervening genes (Fig. 2A).
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Amino acid sequence comparisons showed that the Arabidopsis SUMOs are 32-86% similar to each other but only 17-25% similar to Arabidopsis Ub and RUB1. When compared with yeast Smt3 and human SUMO-1 (9, 12), the Arabidopsis SUMO family is 31-54% similar. Phylogenetic analysis clustered the eight full-length Arabidopsis SUMO proteins into five subfamilies: SUMO1/2, SUMO3, SUMO5, SUMO4/6, and SUMO7/8 (Fig. 2B). Given that SUM4/6 and SUM7/8 genes are arranged in tandem, it is likely these pairs arose from regional duplications of the respective chromosomes. Clustal analysis failed to group any of the Arabidopsis SUMO proteins with animal and yeast versions (Fig. 2B). For example, SUMO1 is equally related to human SUMO-1, -2, and -3 (42, 42, and 40% similar, respectively). This prevented the assignment of orthologous polypeptides and suggested that the Arabidopsis subfamilies arose after the evolutionary split of the animal, fungal, and plant kingdoms.
Sequence alignments of an array of Arabidopsis, human, Drosophila, and yeast SUMO proteins showed that the central region is most conserved (Fig. 2C). This region encompasses the "Ub fold" recently identified as a three-dimensional structure common among plant and animal Ubs, yeast RUB1, rat SUMO-1, and the autophagy polypeptide tag APG8 (or GATE-16) (8, 40-42). As a result, it is likely that Arabidopsis SUMOs assume a similar shape to rat SUMO-1 despite substantial sequence divergence. As with yeast and animal SUMOs, Arabidopsis SUMO1-8 bear additional C-terminal residues beyond the glycine necessary for conjugation (Fig. 2C). Presumably, these residues are removed post-translationally by SUMO proteases to generate the mature active tags of 91-106 amino acids. Whereas most mature Arabidopsis SUMOs, like Ub and RUB1, are predicted to terminate in a glycine-glycine motif, three are predicted to end in a single glycine (SUMO4, -6, and -7) (Fig. 2C). Whether this distinction affects SUMO4/6/7 processing and/or activity is not yet known.
The most divergent region among SUMOs encompasses the N-terminal 14-30
amino acids just proximal to the Ub fold. Structural analysis of rat
SUMO-1 suggests that this hydrophilic region assumes a flexible
solvent-exposed domain that extends from the body of the protein (8).
Although its function is unknown, one possibility is that this
extension imparts unique properties/functions to the individual members
of the SUMO family. Consistent with this notion, we identified the
consensus SUMO attachment site (KXE (2)) within the
N-terminal extensions of Arabidopsis SUMO4 and -6, in each
case represented by the same VKME sequence (Fig. 2C). Its
presence suggests that both SUMO4 and -6 can form poly-SUMO chains,
using the lysine as the connection site (9).
We detected transcripts for four of the eight intact Arabidopsis SUM loci. SUM1, SUM2, and SUM5 cDNAs are in the Arabidopsis EST data bases (www.Arabidopsis.org/Blast), and an mRNA for SUM3 was identified by RT-PCR of total RNA from young seedlings. RNA gel blot analyses of SUM1 and SUM2 showed that both genes are expressed in light- and dark-grown seedlings and in all organs tested, indicating the corresponding proteins are present in most Arabidopsis tissues/cells (data not shown). Both the analyses of ESTs and the signal strength on RNA blots suggested that mRNA levels for SUM1 is higher than SUM2 (Table I and data not shown). We could not detect mRNAs for SUM4 and SUM6-8 either by RT-PCR or RNA gel blot analysis of total RNA isolated from 6-day-old seedlings, suggesting that they are either not expressed, expressed at levels below detection, or that their expression is confined to specific cell types or developmental stages not examined here.
The SUMO Conjugation Pathway Is Present in Arabidopsis--
To
confirm that the Arabidopsis SUMOs become attached to other
proteins, we used immunoblot analysis with anti-SUMO1 and -3 antibodies
to detect the free and conjugated forms in crude extracts. The
antibodies were generated against bacterially expressed antigens containing all residues but those following the C-terminal glycine (Fig. 2C). The anti-SUMO3 antibodies showed a slight
cross-reaction with SUMO1, its closest relative by phylogenic analysis
(Fig. 3A). In contrast, the
anti-SUMO1 antibodies appeared to be specific to the SUMO1/2 group; it
easily detected SUMO1 and its closest relative SUMO2 (83% identity)
expressed in planta but failed to detect SUMO3 (42%
identity) (Fig. 3A and below). Although not tested, we
consider it unlikely that the anti-SUMO1 and -3 antibodies recognize
SUMO4-8 given their even greater phylogenic distance from these two
antigens.
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When both SUMO antibodies were used to probe crude
Arabidopsis extracts, an array of immunoreactive proteins
was evident (Fig. 3A). The anti-SUMO1 antibody recognized an
abundant species of 14 kDa that co-migrated with recombinant SUMO1;
this species likely represents free SUMO1 and -2. In addition, a
heterogeneous smear of high apparent molecular mass proteins, with many
larger than 80 kDa, was also detected that presumably represent SUMO1/2
conjugates. A similar diverse array of conjugates was also detected in
other plant species, including corn, oats, pea, tomato, tobacco, and alfalfa (data not shown). Consistent with studies using yeast, animal,
and tomato cells (2, 20, 21, 25), we detected by confocal microscopic
immunolocalization SUMO1/2 in the cytoplasmic and nuclear compartments
of Arabidopsis cells. As shown in Fig. 4 for heart stage embryos, both punctate
staining coincident with DNA staining within the nucleus and more
diffuse staining of the cytoplasm were evident that could not be seen
with preimmune antiserum (data not shown). Following centrifugal
separations of crude extracts, conjugates could be detected by the
anti-SUMO1 antibodies in the soluble and particulate fractions,
indicating that both soluble and membrane-associated proteins are
sumolated by SUMO1/2.
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In contrast, the anti-SUMO3 antibodies detected little free SUMO and a less complex assortment of SUMO conjugates with few larger than 80 kDa (Fig. 3A). The dominant species were 18, 21, 47, 52, 80, and 150 kDa with only a trace at the predicted migration position of free SUMO3 (14 kDa). Surprisingly, only one species of 48 kDa was in common with those detected by the anti-SUMO1/2 antibodies. Whether this signal represents cross-reaction of the antibodies with a non-sumolated protein or a target modified by both SUMO1/2 and -3 remains to be determined. Although it is possible that the weak signal at 15 kDa represents free SUMO3, given that the anti-SUMO3 antibodies can recognize SUMO1 (albeit poorly) and that free SUMO1/2 is abundant in Arabidopsis extracts, this signal more likely represents free SUMO1/2. Overall, differences in the levels of their free forms and the distinct SDS-PAGE profiles of their conjugates revealed that SUMO1/2 and SUMO3 have different conjugation dynamics and modify distinct sets of Arabidopsis targets.
To test if we could recapitulate this sumolation in vitro, 32P-labeled SUMO1 was added to crude plant extracts under conditions that promote SUMO conjugation (+ATP). In addition to Arabidopsis, wheat germ was tested based on previous studies (38) demonstrating that it represents a rich source of in vitro ubiquitination activity. By using full-length unprocessed SUMO1, a heterogeneous profile of SUMO1 conjugates was synthesized in both extracts by an ATP-dependent reaction (Fig. 3B). Because conjugation of this SUMO should first require trimming by SUMO proteases to remove the C-terminal SGGGATA extension (see Fig. 2C), this activity implies that one or more of these proteases was working in the extracts as well.
Identification of Enzymes Required for SUMO Processing,
Conjugation, and Disassembly--
Like ubiquitination, sumolation
requires a number of accessory proteins as follows: (i) for processing
the SUMO precursors, (ii) for the ATP-dependent multistep
enzymatic cascade to attach SUMOs to various targets, and (iii) for
ultimately removing the bound SUMOs (see Fig. 1). By using yeast and
animal genes as queries, we identified by Blast one or more
Arabidopsis loci predicted to encode each of these required
catalytic steps (Fig. 5).
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In animals and yeasts, the E1 responsible for ATP-dependent SUMO activation is a heterodimer, structurally and functionally related to the single E1 (or UBA1) polypeptide required for Ub activation (Fig. 5A). The SUMO Activating Enzyme (SAE)-1 subunit corresponds to the N-terminal half of UBA1 and likewise contains a Thiamine Biosynthesis Protein F (ThiF) family domain present in members of the ThiF/MeoB/HesA family (Pfam accession number PF00899). The SAE2 subunit corresponds to the C-terminal part of UBA1. It contains a second ThiF domain with a GXGXXG motif that likely participates in ATP binding. This domain is followed by a positionally conserved cysteine in the consensus KXXP(V/G)CTXXXXP motif (29) that activates SUMOs by forming the ATP-dependent thiol-ester linkage with the tag. In addition, SAE2 has a UBA- C- terminal (UBACT) domain (Pfam accession number PF02134). This domain of unknown function is also present in Ub-activating enzymes and is located downstream of the active-site cysteine. Our search identified two Arabidopsis genes encoding SAE1 proteins of 81% amino acid sequence identity to each other and 28 and 32% identity to their Drosophila and humans orthologs, respectively (Fig. 5A). The presence of ESTs and RNA gel blot analyses indicate that both SAE1a and SAE1b are expressed (Table I and data not shown). Only one gene was detected that encodes the second subunit, SAE2 (Fig. 5A). It is 28-33% identical to its animal orthologs and likewise contains the active-site cysteine, the UBA-CT domain, and a C-terminal motif (amino acids 646-650) proposed to function as a nuclear localization signal. SAE2 is expressed, but both the scarcity of ESTs and weak signals by RNA gel blot analysis suggest that its expression is low as compared with SAE1a and -b (Table I and data not shown).
Following activation, SUMOs are transferred to the SUMO-conjugating enzyme (E2), identified as UBC9 in yeast (10, 11). We identified two Arabidopsis loci encoding related proteins designated here as SUMO Conjugating Enzyme (SCE)-1a and -b. SCE1a encompasses the entire E2, whereas SCE1b appears truncated, missing 53 residues from the N-terminal end (Fig. 5A). Like UBC9, SCE1a belongs to a family of conjugating enzymes, all of which have a conserved UBCC (Ub- Conjugating enzyme Catalytic) domain that spans most of the protein and includes a positionally conserved cysteine that forms the thiol ester linkage with the peptide modifier. SCEs are the most conserved across eukaryotes as compared with other components of the sumolation pathway; for example Arabidopsis SCE1a is 53% identical to its yeast ortholog UBC9. Both the presence of ESTs and RNA gel blot analysis indicate that SCE1a is highly expressed in many Arabidopsis tissues (Table I and data not shown). Anti-SCE1 antibodies also detected the resulting protein in roots, leaves, flowers, and stems of soil-grown plants (data not shown). We have been unable to detect the SCE1b transcript by RNA gel blot analysis and RT-PCR or the predicted SCE1b protein by immunoblot analysis. Coupled with the apparent truncation of its coding sequence, we consider it likely that SCE1b is a pseudogene.
In the Ub conjugation pathway, target recognition and Ub transfer ultimately require a Ub ligase or E3 (5, 6). In Arabidopsis, a multitude of E3s exist, suggesting a one-to-one correspondence to individual targets (43). Recently, a small family of SUMO E3s has been identified in both yeast and animals (13, 30, 44, 45, 47). They contain a RING-like domain called Siz-PIAS RING (SP-RING), which may facilitate interactions with UBC9, and a chromatin organization domain SAF-A/B-Acinus-PIAS (SAP). Thus far, we could detect only a single potential SUMO E3 ortholog in Arabidopsis (AtSIZ1) bearing both the Siz-PIAS and SAP motifs (Fig. 5A). A number of AtSIZ1 ESTs are evident, suggesting that this gene is highly expressed (Table I).
SUMO proteases specifically remove amino acids distal to the C-terminal glycine important for conjugation and thus are essential for processing SUMO precursors to generate the active polypeptides and for releasing bound SUMOs to regenerate targets and SUMOs in unmodified forms (2, 4) (Fig. 1). Two types of SUMO proteases have been described thus far in animals related to yeast Ub-Like Protease-1 (ULP1) and ULP2/Smt4 (31, 32). Both types contain a conserved 200-amino acid ULP1-Catalytic (ULP1-C) domain that surrounds a catalytic triad of histidine, aspartate, and cysteine residues (31, 32). Searches of the Arabidopsis database identified 12 genes (designated here as ULP1a-d and ULP2a-h) that encode the consensus UBP1-C domain with the His/Asp/Cys catalytic triad (Table I and Fig. 5, A and B). Phylogenic analysis of the ULP1-C domain clustered the 12 into three subfamilies with two subfamilies more related to yeast ULP1 and the third more related to yeast ULP2/Smt4 (Fig. 5B). Outside of the ULP1-C domain, the Arabidopsis ULPs bear little sequence similarity, suggesting that they attack different targets. In addition, we detected a number of ULP-like proteins with the ULP1-C signature but missing one or more of the essential amino acids that form the catalytic triad (data not shown). Whether these are functional ULPs is not yet known.
Stress-induced Accumulation of SUMO Conjugates--
Given the
observations that sumolation is activated by stress and that some SUMO
targets are part of the stress response in animals (2, 20-23), we
tested if SUMOs play a comparable role in plants. Here, we subjected
young Arabidopsis seedlings grown in liquid culture to
various stress conditions, and we measured the levels of free and
conjugated SUMO1/2 and SUMO3 by immunoblot analysis of crude seedling
extracts. Stress conditions included exposure to heat (28-47 °C)
and cold shock (4 °C), high NaCl (75-300 mM) or
osmoticum (75 mM to 1 M mannitol and sorbitol),
toxic metals (0.2-100 mg/liter lead, copper, cadmium, cesium, and
arsenic), 7% ethanol, the amino acid analog canavanine (0.3-30
mg/liter), and oxidative stress elicited by paraquat (0.2-20
mM) or H2O2 (5-50 mM)
(Figs. 6 and
7 and data not shown).
|
|
Although many stress conditions had little or no effect on the pools of free and conjugated SUMO1/2 and -3, exposure to heat, H2O2, ethanol, and canavanine induced a dramatic increase in SUMO conjugates that was specific for the SUMO1/2 isoforms (Figs. 6 and 7). For example within 30 min from the beginning of a temperature shift from 24 to 37 °C, a dramatic rise in SUMO1/2 conjugates was evident as compared with PBA1, a 26 S proteasome subunit used to verify equal protein loads (33) (Fig. 6). This increase preceded the accumulation of HSP101, a chaperone whose synthesis is rapidly up-regulated following heat shock (48), suggesting that the response is fast. From quantitations of the blots, we estimated that the levels of SUMO1/2 conjugates increased ~6-fold during the heat stress. In contrast, the levels of SUMO3 conjugates remained constant during the heat stress, whereas the levels of Ub conjugates, previously shown to accumulate during heat stress (49, 50), displayed a slower and more modest increase (Fig. 6).
Concomitant with the rise in SUMO1/2 conjugates was a significant drop in the levels of the free form raising the possibility that free SUMO1/2 became limiting during the heat stress. To examine this possibility and prove that SUMO2 is also involved in the response, we tested transgenic Arabidopsis lines overexpressing SUMO2 under the control of the 35S promoter. As can be seen in Fig. 6, these lines had increased levels of free and conjugated SUMO1/2 under non-stressed conditions. When subjected to heat shock, the levels of SUMO1/2 conjugates rose even further to levels beyond that seen for wild-type plants. Only a modest drop in free SUMO1/2 was observed, suggesting that these transgenic plants were now saturated for the free forms. Thus under acute heat stress, free SUMO1/2 and not their targets, appears to be limiting in wild-type plants. Despite such changes, the phenotype of the 35S-SUMO2 plants were indistinguishable from wild type when grown under a variety of conditions including elevated temperatures (data not shown). They also showed no change in the induction of HSP101 accumulation during heat stress, suggesting that the heat shock response was not altered as well (Fig. 6).
In a similar fashion to heat shock, exposure to 7% ethanol or increasing concentrations of H2O2 and canavanine dramatically increased the levels of SUMO1/2 conjugates (Fig. 7). The effects of ethanol and H2O2 were rapid, with SUMO1/2 conjugates rising within 10 min after their addition to the culture medium (data not shown). In contrast, the effect of canavanine was much slower, requiring hours to elicit a response. This slower response is consistent with the fact that this amino acid analog must be taken up by the plant and translationally incorporated into protein before inflicting cellular damage. Like heat shock, these stresses substantially changed the amount but not the profile of SUMO1/2 conjugates, suggesting that the same array of targets was sumolated with the stress simply increasing the percentage in the modified form. The only major difference among the four stress treatments was an abundant 31-kDa conjugate that specifically appeared during a 24-h exposure to canavanine (Fig. 7). In contrast to heat shock, we did not observe a drop in free SUMO1/2 suggesting that the other stresses consumed less of the free pool when forming conjugates. Whereas the ethanol and H2O2 treatments failed to induce the expression of HSP101 within the 60 min that we observed an increase in SUMO1/2 conjugates, an increase in HSP101 levels was evident following a 24-h exposure to canavanine.
To examine the heat-stress response in more detail, we varied the
duration and magnitude of the treatment and examined their effects on
the accumulation of SUMO1/2 conjugates. As shown in Fig.
8 using increasing lengths of the heat
shock, the response is transient with the duration dependent on pulse
length. A 2-min incubation of the cultures at 37 °C induced only a
mild accumulation of SUMO1/2 conjugates that subsequently returned to
normal levels ~30 min later. Progressively longer incubations
increased not only the amount of conjugates but also their duration
such that a 120-min exposure to 37 °C generated an increased pool of
SUMO1/2 conjugates that remained in the seedlings for more than 2 h after their return to 22 °C. The rise and fall of SUMO1/2
conjugates were paralleled by a decline and reappearance of free
SUMO1/2 (Fig. 8). This inverse relationship suggested that the SUMO1/2 modifier was not consumed following conjugation but was instead released from sumolated proteins after the heat shock was over. Like
other heat shock responses (48, 51), the Arabidopsis seedlings required temperatures 34 °C to induce a significant increase in SUMO1/2 conjugates with the maximal response at 40 °C
(Fig. 9). The response was also
remarkably rapid. We detected a rise in SUMO1/2 conjugates within 2 min
after transferring the cultures to the 40 °C water bath (Fig. 9),
which was even more impressive when considering the fact that the
medium needed almost 8 min to reach this heat shock temperature under
our experimental conditions.
|
|
Surprisingly, heat-induced sumolation appeared to adapt to the stress.
Plants subjected to a first heat treatment at 37 °C showed a
substantial but transient accumulation of SUMO1/2 conjugates (Fig.
10). However, only a slight increase in
conjugates was evident for a second heat shock if given within 5 h
of the first. This dampened response required almost 20 h to fully
dissipate. A comparable effect for the HSP101 protein was not observed.
Instead HSP101 accumulated following the first heat shock and remained
at high levels for over 24 h with or without a second heat
treatment (Fig. 10).
|
The rapid and temperature-dependent nature of the response
along with its transient effect and adaptability implied that
stress-induced sumolation by SUMO1/2 has an important regulatory role
in the plant stress response. To help confirm this role, we examined the SUMO1/2 conjugate patterns in Arabidopsis lines
constitutively expressing high levels of a cytosolic isoform of HSP70,
designated HSC70-1.2 The HSC70-1 gene is
expressed in most Arabidopsis tissues and is mildly
up-regulated by heat shock (52). Similar to the behavior of comparable
Drosophila and mammalian mutants (53, 54), these HSC70-1-overexpressing lines are more thermotolerant, exhibiting an
increased survival to an acute 44 °C heat shock.2 Young
homozygous HSC70-OX seedlings contained 3-4 times more HSC70 protein
as compared with wild type or a weak expresser but normal levels of
total protein and a variety control proteins such a PBA1 (Fig.
11 and data not shown). When subjected
to a 37 °C heat stress for 30 min, these overexpressing seedlings
accumulated substantially less HSP101 protein, presumably caused by a
lower demand for other types of chaperones. In addition, these HSP70-OX plants accumulated fewer SUMO1/2 conjugates during the heat shock and
concomitantly retained more of the free forms (Fig. 11). Both the peak
amount of SUMO1/2 conjugates and their duration within the plants were
dampened (e.g. compare levels at the 120-min time point),
suggesting that the accumulation of SUMO1/2 conjugates is
physiologically relevant to thermotolerance.
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DISCUSSION |
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In addition to Ub, it is now becoming clear that plants use a variety of other polypeptide tags to post-translationally modify and thus regulate numerous intracellular proteins (1, 55). Here we show that the family of SUMO proteins recently discovered in yeast and animals represents another class of influential modifiers. Through searches of the Arabidopsis genome databases, we discovered loci predicted to encode all components of the SUMO modification system, including the SUMO tag and the various steps required for SUMO processing, conjugation, and release. In total, 25 Arabidopsis loci were identified that appear functional (Table I) as follows: 8 genes encoding SUMO, 3 for the E1, 1 for the E2, at least 1 potential E3, and a 12-gene family encoding putative SUMO proteases. The similarity of these protein sequences to those found in yeast and animals indicates that the SUMO pathway has been strongly conserved during eukaryotic evolution. However, the larger size of this gene collection in Arabidopsis suggests that plants in particular have expanded further upon the use of SUMO conjugation and possibly added new functions to this protein modification system.
Of the nine Arabidopsis SUM loci, eight (SUM1-8)
are predicted to encode the expected full-length proteins of 94-103
amino acids, making this family the largest known so far in a
eukaryote. By comparison, yeast encodes a single SUMO (Smt3), whereas
humans encode only three, SUMO-1, -2, and -3 (23). Like their yeast and
animal counterparts, the Arabidopsis proteins bear a long N-terminal region extending beyond the Ub fold and terminate in a 4-14
amino acid sequence that caps the C-terminal glycine essential for
attachment. Two (SUMO4 and -6) have the consensus sumolation site
(KXE) near their N termini, suggesting they may be
involved in forming polymeric SUMO chains (9). Expression studies
indicate that at least four of the Arabidopsis SUM genes are
transcribed, implying that a complex assortment of SUMO isoforms may
exist in each cell type. In contrast, SUM9 appears to be a
pseudogene. Such pseudogenes have been described in other organisms;
for example, there are three predicted SUMO-1 pseudogenes in
humans and two in mice (56).
The SUMO pathway biochemically resembles the more thoroughly studied Ub pathway (5, 6). Structurally, the most prominent difference between these two pathways is the heterodimeric nature of the SUMO E1 (2, 57). Like its yeast and mammalian orthologs, the Arabidopsis SUMO E1 subunits SAE1 and SAE2 show similarity to N- and C-terminal parts of the monomeric E1 in the Ub pathway (Fig. 5A). In contrast to all other organisms studied to date where both SAE subunits are encoded by a single gene, the Arabidopsis SAE1 subunit is encoded by two highly similar genes. Given that SAE1 could have a regulatory role by influencing the active site in SAE2, the use of two distinct isoforms could provide a way to modulate sumolation or selectively activate specific SUMO isoforms. Clearly, analyses of tissue-, cell cycle-, or stress-related expression patterns of the SUM and SAE1 genes and enzymatic characterizations of the resulting proteins are needed. The ULPs, presumably involved in SUMO processing and/or release, are the largest collection of isozymes in Arabidopsis. The size of this group argues that de-sumolation must be an important step in SUMO function and regulation in plants, possibly by creating a reversible modification cycle akin to phosphorylation/dephosphorylation (4, 58).
Previous studies with human SUMOs (9, 23) and our phylogenic analysis of the Arabidopsis SUMOs suggested that several functionally distinct isoforms exist. Our immunological studies of Arabidopsis SUMO1/2 and -3 support this notion by showing that each is conjugated to a distinct set of targets. SUMO3 becomes attached to only a few proteins with a range of molecular masses with little of the free form evident. In contrast, a substantial percentage of SUMO1/2 exists in the free form with the remaining present in a heterogeneous collection of conjugates with masses >80 kDa. Whereas the levels and distribution of SUMO3 conjugates appear unaffected by stress, the levels of SUMO1/2 conjugates are dramatically but transiently increased by various types of cellular stress, including exposure of the plants to heat shock, H2O2, ethanol, and canavanine. The increase and decrease in conjugates following heat shock are paralleled by a substantial decrease and increase in the free SUMO1/2 pool, suggesting that the heat-induced conjugation is reversible. The remarkable feature of this response is its speed, occurring within minutes of heat shock, thus implicating a conjugation cascade that can rapidly respond to stress signals.
Because several types of stress elicit the same accumulation of SUMO1/2 conjugates, it is likely that a more general outcome, such as the accumulation of denatured/damaged proteins, rather than the stimulus is the signal. A role for damaged proteins is consistent with our studies with Arabidopsis plants engineered to constitutively express HSC70-1, a cytosolic version of HSP70 that helps refold damaged proteins (52, 59).2 Similar to mammalian cells and Drosophila overexpressing Hsp70 (53, 54), these plants have increased thermotolerance presumably due to their increased capacity to repair damaged proteins that accumulate during heat stress. Here, we found that such HSC70-OX lines also have a dampened accumulation of SUMO1/2 conjugates, implying that this accumulation is triggered either directly or indirectly by damaged proteins. It is tempting to speculate that signaling pathway(s) initiated by the accumulation of unfolded proteins (e.g. the unfolded protein response (60)) directly activates sumolation.
Despite the lack of obvious amino acid sequence relationships, both the profiles of the free and conjugated forms and their response to stress argue that Arabidopsis SUMO1/2 and SUMO3 are functionally analogous to human SUMO-2/3 and SUMO-1, respectively (9, 23). Like that for Arabidopsis SUMO3, the SDS-PAGE profile of human SUMO-1 conjugates showed that little is in the free form with most conjugated to a limited set of targets whose abundance is unaffected by stress (23). One of the main targets of animal SUMO-1 is RanGAP (14). Consistent with this potential similarity, we detected an Arabidopsis SUMO3 conjugate of near 80 kDa that matches the size of Arabidopsis RanGAP plus one SUMO3. However, it should be noted that the recent cloning of plant RanGAP genes revealed that the encoded proteins are missing the consensus sumolation domain found in their animal counterparts (61). In contrast, the SDS-PAGE profile of human SUMO-2/3 conjugates, like that for Arabidopsis SUMO1/2, showed that a substantial percentage is in the free form with the rest present in a heterogeneous array of conjugates. The levels of these conjugates are dramatically increased by various stresses, including exposure of the cell cultures to heat shock, ethanol, H2O2, N-ethylmaleimide, osmotic stress, and DNA-damaging agents such as UV light and cisplatin (22, 23). The only notable difference between Arabidopsis SUMO1/2 and human SUMO-2/3 is the absence of a consensus sumolation site in the Arabidopsis forms.
The differential actions of SUMO1/2 and -3 raise the possibility that
the other Arabidopsis SUMOs also have unique functions. Based on phylogenic clustering predictions alone, this plant has five
distinct SUMO types (SUMO1/2, -3, -4/6, -5, and -7/8 (Fig. 2B)) that each could become attached to a different set of
targets. For the clade comprising SUMO4 and -6, one obvious possibility based on the presence of the consensus sumolation site
(KXE) is that this type participates in forming poly-SUMO
chains. As a result sumolation could have a number of diverse roles in
plant cell regulation beyond that seen here for SUMO1/2 and -3. How would each of these SUMO isoforms become preferentially attached to
their respective targets? To date, we identified two types of E1s
(based on use of the SAE1a and -b isoforms), only one functional E2
(SCE1), and only one E3 (SIZ1). Given that this small number seems
insufficient to attach selectively the various SUMO isoforms, it is
likely that additional activities are necessary, especially with
respect to the E3 activities required for ligation. One additional candidate for an E3 could be an Arabidopsis ortholog of
RanBP2 which appears to be a SUMO-1 ligase in mammalian cells (57).
Why are Arabidopsis SUMO1/2 conjugates and their potential counterparts in animals increased upon stress? The transient nature of the increase in Arabidopsis and the fact that the free SUMO1/2 pool recovers after the stress imply that SUMO1/2 is in a dynamic equilibrium between the free and conjugated forms and that the SUMO moiety is not consumed during the cycle. That the levels of conjugates under both non-stressed and stressed conditions can be elevated by overexpressing SUMO2 indicates that the pool size of free SUMO1/2 is a major limitation in forming these conjugates. An intriguing aspect of this heat-induced sumolation is our observation that the plants adapt transiently to the stress. Whereas the response is robust initially, subsequent heat shocks are less effective if given shortly after the first, despite an apparently adequate supply of free SUMO1/2. This dampening implies that either the SUMO conjugation system is attenuated and/or that the availability/abundance of targets is substantially reduced following the first heat stress. Under our experimental conditions, almost 20 h were needed to fully reactivate the response and/or replenish the substrates.
One possibility is that SUMO1/2 conjugation plays a regulatory role in the stress response. Here attachment of SUMO1/2 could alter the activity or location of critical effector(s) of the stress response. Consistent with this possibility are the facts that a majority of Arabidopsis SUMO1/2 (Fig. 4) and the heat up-regulated SUMO conjugates in mammalian cells (22) are nuclear localized and that a number of nuclear DNA-binding proteins in mammalian cells such as p53, c-Jun, c-Myb, AP-2, and the androgen receptor are SUMO targets (15-19). In several cases this sumolation appears to alter the association of these factors with cognate DNA-binding sites, either positively or negatively depending on the target. Two notable SUMO targets are HSF1 and -2, which are responsible for up-regulating many HSP genes during heat shock and other stresses (20, 21). Importantly, sumolation of these proteins increases their affinity for heat shock DNA elements. Whereas HSF2 is constitutively sumolated, the level of HSF1-SUMO conjugates is dramatically up-regulated by heat shock supporting this modification as an activator of HSF1 function (20, 21).
In a similar fashion, it is possible that the SUMO1/2 conjugation in Arabidopsis helps activate (deactivate) a battery of nuclear regulatory proteins whose activities are needed at low (high) levels in non-stress plants and at high (low) levels when the plants are exposed to various stress signals. Potential targets under negative regulation could include a battery of factors that promote cell division and other general physiological processes that should be repressed as plants cope with adverse environments. Potential targets under positive regulation could include the 21 HSF-like proteins present in the Arabidopsis genome (www.uni-frankfurt.de/fb15/botanik/AFGNnvr.html) whose activation promotes the stress response (46). Consistent with this notion, we showed here that overexpression of HSC70, which should lessen demand for the targets of HSFs, reduces the heat-induced accumulation of SUMO1/2 conjugates. Such a regulatory role is also supported by the observations that plant defense responses to pathogens may involve SUMO conjugation (25, 26). In particular, one can imagine that AvrBsT from the plant pathogen X. campestris, which may act as a SUMO protease (26), interferes with the stress response that attenuates pathogen invasion by de-sumolating and thus deactivating an important stress regulatory factor. For other targets in animals, sumolation appears to control cytoplasmic/nuclear partitioning and location within the nucleus (2-4). For example, sumolation of RanGAP helps regulate the nuclear pore, whereas sumolation of Sp100 promotes its association with promyelocytic leukemia bodies, nuclear foci of unknown function. Thus, stress-induced sumolation could also affect the activity of various plant regulatory proteins by controlling their transport into or out of the nucleus or their association with other nuclear components.
Another possibility is that stress-induced sumolation plays a more
direct role in the stress response by protecting individual proteins
from unfavorable conditions (23). In a similar fashion to the
antagonistic role of sumolation in Ub-mediated IB
degradation (24), heat-induced sumolation could block the turnover of proteins by
masking their Ub attachment sites. These targets could be regulatory proteins whose stabilization is needed to elicit a robust stress response or could be unfolded proteins destined for repair after relief
from the stress condition. Alternatively, SUMO once attached could by
itself protect native proteins from unfolding or stabilize unfolded
proteins from aggregation or further denaturation and thus work
cooperatively with proteins like HSC70. Such a mass action is supported
by our observations that the level of SUMO1/2 and not the targets
appear to be limiting during heat shock. Given the dynamic nature of
the SUMO tag, it is also possible that SUMO1/2 actually promotes
degradation. Here, SUMO1/2 conjugation could increase the solubility of
denatured proteins formed during stress and thus enhance their removal
by proteolytic pathways such as the Ub/26 S proteasome system.
Whatever their role, an increase in SUMO1/2 conjugates is by itself
insufficient to enhance stress tolerance in Arabidopsis.
Despite the fact that transgenic plants overexpressing SUMO2 had
increased levels of free SUMO2 and SUMO1/2 conjugates, they exhibited
the same sensitivity to heat shock as wild-type plants by several
diagnostic parameters (51), including reduced hypocotyl growth and
cotyledon expansion and increased chlorosis (data not shown).
Collectively our data point to sumolation as an important
post-translational modification in plants that may have a number of
roles created by the use of distinct SUMO isoforms. In
Arabidopsis, it is clear that an array of proteins become
modified by this conjugation system. The rapid and reversible formation
of SUMO1/2 conjugations following exposure of seedlings to stress
points to sumolation as an important regulator of the stress response. Determining how these stresses activate the conjugation cascade and
identifying the various targets will be paramount to defining how
sumolation ultimately participates in stress protection and/or recovery.
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ACKNOWLEDGEMENTS |
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We thank Drs. Elizabeth Vierling for anti-HSP101 antibodies, Charles Guy for the HSC70-OX line and the HSC70 antibody, Anita Fernandez for assistance with the immunolocalizations, and Jed Doelling for helpful discussions.
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FOOTNOTES |
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* This work was supported by United States Department of Agriculture-National Research Initiative Competitive Grants Program Grants 97-35301-4218 and 00-35301-9040, the Research Division of the University of Wisconsin College of Agriculture and Life Sciences Grant Hatch 142-N936 (to R. D. V.), a NATO Research Fellowship (to J. S.), and a United States Department of Agriculture-National Research Initiative Competitive Grants Program Grant 00-35100-9532 (to Dr. Charles Guy to support D.-Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF510519-AF510525.
§ Both authors contributed equally to this work.
¶ Present address: Plant Developmental Biology, Max-Planck-Institute for Plant Breeding Research, 50829 Cologne, Germany.
** To whom correspondences should be addressed: Dept. of Horticulture, University of Wisconsin, 1575 Linden Dr., Madison, WI 53706. Tel.: 608-262-8215; Fax: 608-262-4743; E-mail: vierstra@facstaff.wisc.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M209694200
2 D.-Y. Sung and C. L. Guy, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: E1, SUMO-activating enzyme; E2, SUMO-conjugating enzyme; E3, SUMO ligase; Ub, ubiquitin; SUMO, small ubiquitin-like modifier; EST, expressed sequence tag; RT, reverse transcribed; HSP, heat shock protein; HSC, heat shock cytosolic protein; HSF, heat shock transcription factor.
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