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TRENDS in Plant Science
Vol.6 No.10 October 2001
http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02080-5
463
Review
Andreas Bachmair
Institute of Botany,
University of Vienna,
Rennweg 14, A-1030
Vienna, Austria.
e-mail:
bachmair@s1.botanik.
univie.ac.at
Maria Novatchkova
Frank Eisenhaber
Research Institute of
Molecular Pathology, Dr.
Bohrgasse 9, A-1030
Vienna, Austria.
Thomas Potuschak
Institute of Cellular and
Molecular Biology,
University of Edinburgh,
Mayfield Road,
Edinburgh, UK EH9 3JR.
Covalent addition of ubiquitin to substrate proteins is
apparently widely used in plants, to such an extent
that it is difficult to name any biological process
without direct or indirect connection to a
ubiquitylation step. Ubiquitylation of substrate
proteins involves the following steps
1
(Fig. 1):
activation of ubiquitin’s C-terminal Gly by linkage
to a Cys residue of ubiquitin-activating enzyme (E1),
the transfer of ubiquityl moieties to a Cys residue
in a ubiquitin-conjugating enzyme (E2), and the
final formation of an isopeptide bond to a Lys residue
of a substrate with the help of a ubiquitin–protein
ligase (E3).
The linkage of additional ubiquitin moieties
frequently follows transfer of the first ubiquitin
moiety to the ubiquitylated substrate.
Ubiquitylation can have a diverse range of
consequences for the substrate protein, depending
largely on the type of linkage to ubiquitin. The most
prominent examples are the degradation of
ubiquitylated proteins by the proteasome or in the
vacuole. In this article, we restrict our survey to
the steps necessary to link ubiquitin to substrates
and give an overview of the proteins involved in
these steps. Recent reviews provide additional
information
2–6
.
Ubiquitin
Ubiquitin genes of Arabidopsis(Fig. 2) have been
characterized extensively
7–10
. As in other eukaryotes,
all these genes encode precursor proteins, frequently
in a head-to-tail arrangement of several ubiquitin
moieties; the alternative arrangement is a fusion of
ubiquitin to one of two ribosomal proteins. The
precursors need proteolytic processing by ubiquitin-
specific proteases to release mature ubiquitin. For the
polyubiquitin genes UBQ8, UBQ9, UBQ12and
UBQ13, there are no corresponding EST sequences,
indicating that these genes are not, or are only poorly,
transcribed. Incidentally, these presumed
pseudogenes are also less well conserved at the
protein level. In the genes UBQ7and UBQ15, which
have no
ORTHOLOGS (see Glossary) in Saccharomyces
cerevisiae, a single ubiquitin moiety is fused in frame
to the ubiquitin-like protein
RUB (related to ubiquitin).
The existence of these UBQ–RUBfusion genes in
Arabidopsisis suggestive of functional
interdigitation
11,12
. Ubiquitin-like proteins can indeed
function as regulators of ubiquitylation.
In this article,we summarize
Arabidopsis
genes encoding ubiquitin,ubiquitin-
activating enzyme (E1), ubiquitin-conjugating enzymes (E2s) and an
additional selected set of proteins related to ubiquitylation.We emphasize
comparisons to components from
Saccharomyces cerevisiae
,with
occasional reference to animals.Among the E1 and E2s,
Arabidopsis
usually
has two to four probable orthologs to one yeast gene. Also,
Arabidopsis
has
genes with no likely ortholog in yeast, although they often have potential
orthologs in animals.The large number of components with known
function in ubiquitylation indicates that this process plays a complex role in
cellular physiology.
Ubiquitylation in plants:a post-
genomic look at a post-translational
modification
Andreas Bachmair,Maria Novatchkova, Thomas Potuschak and Frank Eisenhaber
E2
E1
E1
E1
E2
SH
S
SH
S
S
HS
E2
E2
TRENDS in Plant Science
E2
SH
S
E3
E3
E3
Fig. 1. Ubiquitin (yellow) is activated by a ubiquitin-activating enzyme,
E1 (red). The free C-terminal Gly of ubiquitin thereby forms a thioester
bond to a Cys residue of E1. Activated ubiquitin is transferred to one of
several ubiquitin-conjugating enzymes, E2 (green), again forming a
thioester. Ubiquitin is then transferred to the substrate (light blue) to
form an isopeptide linkage with an internal Lys residue of the substrate.
Ubiquitylation of substrates usually requires another factor, a
ubiquitin–protein ligase, E3 (indigo), which binds to both E2 and the
substrate. RING-finger-type ligases apparently operate by bringing
substrate and E2 into close proximity, whereas E3 proteins of the
HECT-domain type accept ubiquityl residues from E2 proteins to form
an intermediary thioester before transferring ubiquitin to the substrate.
After ubiquitylation of the substrate, additional ubiquityl residues can
be added onto the already attached ubiquitin. This step can require an
additional factor, E4 (not shown). Which internal Lys of ubiquitin is
chosen for linkage depends on the ligase complex involved.
Ubiquitin-activating enzyme
Ubiquitin activation is not considered to be a step
with a discriminatory or a regulatory role.
Accordingly, both enzymes of Arabidopsis
13
(Fig. 3)
are expected to have the same specificity. A potential
regulatory modification of animal E1 is
phosphorylation, which has been correlated with
subcellular distribution
6
. Because E1 is, at least in
animal cells, located in both the nucleus and the
cytoplasm, ubiquitylation is believed to occur in both
of these cellular compartments.
Ubiquitin-conjugating enzymes
About half of the E2s of Arabidopsis(Fig. 3) have
been characterized biochemically and by isolation of
cDNAs, mostly by Richard Vierstra’s group.
Arabidopsishas at least one close homolog to ten of
the 11 ubiquitin-conjugating enzyme (
UBC) consensus
enzymes of yeast that conjugate ubiquitin (yeast
UBCs were numbered consecutively from 1 to 13;
however, ScUBC9 and ScUBC12 conjugate not
ubiquitin but ubiquitin-like proteins). The 11 yeast
E2s have distinct, although in some cases
overlapping, functions. There is extensive literature
about the biochemical functions and biological roles
of these yeast UBCs [Refs 14,15 and the S. cerevisiae
protein database YPD (http://www.proteome.com/)].
The distinction of different classes of ubiquitin-
conjugating enzymes that are conserved across
phyla is clearly evident in a cumulative evolutionary
tree
16
. Ironically, we could not identify a clear
ortholog for ScUBC3/CDC34, the only essential
yeast E2. The group of ArabidopsisE2s with highest
similarity to ScUBC3 appears to be even more
closely related to ScUBC7, and this group is
therefore listed as likely orthologs of ScUBC7.
Because the role of ScUBC3 in ubiquitylation
depends on
SCF ubiquitin ligases (which play a major
role in plants), other E2s of Arabidopsismust
provide this function.
There are three UBC gene families in
Arabidopsisfor which no probable orthologs exist in
TRENDS in Plant Science
Vol.6 No.10 October 2001
http://plants.trends.com
464
Review
Gene from
Arabidopsis thaliana
ecotype Columbia
AGI
GenBank Accession no.
Gene from
Saccharomyces
cerevisiae
Name
BAC locus
Chr. locus
NT
Protein
Chr. Domain architecture
Ubiquitin extension genes
UBQ1
F22O6.30
AT3g52590
J05507
AAA32904
3
UBI1
,
UBI2
UBQ2
F9C22.10
AT2g36170
J05508
AAA32905
2
UBQ5
T17J13.210
AT3g62250
J05539
AAA32906
3
UBQ6
F14M4.6
AT2g47110
J05540
AAA32907
2
UBI3
UBQ5/6-like
F26F24.5
AT1g23410 AC005292 AAF87001
1
Polyubiquitin genes
UBQ3
F15A17.270
AT5g03240
L05363
CAB86091
5
UBQ4
T1M15.20
AT5g20620
X12853
CAA31331
5
UBQ10
C17L7.240
AT4g05320
L05361
AAA68878
4
UBQ11
T32N4.13
AT4g05050
L05362
CAB81047
4
UBQ14
T5J8.21
AT4g02890
L05394
CAB77774
4
UBQ8
F8A24.16
AT3g09790
L05917
AAA68879
3
UBQ9
K12B20.90
AT5g37640
L05365
S55244
5
UBQ12
T7N22.10
AT1g55060
L05482
NA
1
UBI4
UBQ13
T8F5.13
AT1g65350
L05401
JQ1728
1
UBQ–RUB fusion genes
UBQ15
T19E23.4
AT1g31340 AC007654 AAF24594
1
UBQ7
T20F21 NA L05364 S55242 2
Ribosomal L40
Ribosomal S27
Ubiquitin (UBQ)
UBQ-like
RUB
U
U
U
U
U U U U
U U U U
U U U U
U
U
U U U
U U U
U
U
U
U U U
U
U
U U U U U
U
[
[
6
U
U
U
[
[
4
U
U U U U U
U
U
U
U
U
1
U
U
2
TRENDS in Plant Science
Fig. 2. A list of known ubiquitin genes, excluding ubiquitin-like
proteins and ubiquitin units more distantly related to the canonical
ubiquitin unit. In total, there are 82 ubiquitin-like proteins and proteins
containing ubiquitin-like domains encoded in the
Arabidopsis
genome,
compared with eight in
Saccharomyces cerevisiae
and 38 in humans
(numbers are taken from INTERPRO http://www.ebi.ac.uk/proteome/).
The accession numbers depicted in bold are the sequences that have
been used to describe the domain architecture. In the case of
UBQ10
,
UBQ13
and
UBQ15
, the TAIR protein prediction differs dramatically from
other published protein predictions from the same coding sequence.
UBQ3
,
UBQ4
,
UBQ10
,
UBQ11
and
UBQ14
consist only of UBQ domains
100% identical with that in
UBQ1
.
UBQ3
and
UBQ11
are known to show
intraspecific repeat number diversity (4–6 and 3–6, respectively)
10
.
The GenBank entries U84967 to U84969 reflect allelic
UBQ11
genes of
different size. Two in-frame stops in the first and one in the second
ubiquitin repeat of
UBQ12
, a possible pseudogene, are indicated by
vertical blue lines. The
UBQ13
gene sequence contains an insertion of
mitochondrial DNA in the fifth domain
9
. Abbreviations: Chr.,
chromosome; NA, not available; NT, nucleotide (sequence). The
Arabidopsis
sequence sets of the
Arabidopsis
Genome Initiative (
AGI)
used in this work were obtained from the TAIR website
(http://www.arabidopsis.org/) on March 22nd, 2001.
the budding yeast (Fig. 3). However, all three groups
have potential orthologs in animals. One group is
tentatively called AtUBC15-like (possible human
ortholog BAB14800). The second group has
similarity to the UBC domain of giant UBC
enzymes found in mammals
17
(possible human
ortholog KIAA1734), and these plant proteins are
also unusually large. The third UBC family
that does not exist in yeast has similarity to a
human UBC that was first isolated as an
autoimmune antigen
18
. The existence of these gene
classes is indicative of additional roles for
ubiquitylation in metazoans compared with the
single-celled yeast.
For cases in which Arabidopsishas a gene family
related to a yeast or an animal UBC, we might
expect differences in transcriptional regulation
among family members, and such differences have
been found in, for instance, the ScUBC2- and
ScUBC4-type families
19
. It remains to be seen
whether all members of a family have identical
enzymatic properties.
UBC-domain proteins without the conserved catalytic
Cys residue
Like S. cerevisiae and animals, the Arabidopsis
genome contains several proteins with UBC
similarity that lack the Cys residue that is essential
for catalytic activity (Fig. 4). These proteins are called
ubiquitin-enzyme variants (UEVs). The well studied
proteins of this type have apparently retained the
ability to bind to ubiquitin, although not when
attached to E1 but when linked to
certain substrates. ScMMS2 can form a
heterodimer with ScUBC13, and this dimer has
distinct enzymatic properties, promoting the
formation of the Lys63 ubiquitin–ubiquitin linkage
20
.
In vivo, the ScMMS2–UBC13 dimer and its plant
homologs presumably cooperate with a
ubiquitin–protein ligase. ScVPS23 plays a role after
ubiquitylation, by binding to monoubiquitylated
membrane proteins during vacuolar protein sorting
21
.
Ubiquitin–protein ligases
All known ubiquitin–protein ligases have either a
HECT or a RING-finger domain as a ‘docking site’for
E2. Proteins with a so-called U-box (or UFD-box;
reviewed in Ref. 22) might be related to RING-finger
proteins, because the U-box has a RING-finger-like
fold but lacks the Zn
2+
ions coordinated by RING-
finger domains. Accordingly, one might define two
superfamilies of E3 enzymes. Arabidopsishas
representatives of both categories but their sheer
number precludes a complete listing in this article. A
database search indicates seven HECT domains in
the Arabidopsisgenome and >300 RING-finger
domains. Although it is currently unclear whether all
the RING-finger domains mediate interaction with an
E2 (the domain could, for instance, also mediate
binding to other proteins), it is possible that each
RING-finger protein is part of a distinct
ubiquitin–protein ligase. Table 1 lists the numbers of
selected domains in the Arabidopsis genome and
Fig. 5 shows examples of conserved ligases.
There is a mechanistic difference in the ubiquitin
transfer step between the HECT-domain and the
RING-finger-domain E3 enzymes. HECT-domain
proteins have a Cys residue that forms a thioester
with ubiquitin (transferred to this site from E2),
whereas RING-finger-domain proteins are believed to
be ‘marriage brokers’, with the sole explicit function of
bringing substrate and the E2 enzyme close together.
HECT-domain E3 enzymes characterized to date
seem to be large, often single-subunit proteins. The
RING superfamily also has representatives of single-
subunit enzymes, the most prominent being the
N-
END-RULE E3s such as UBR1 of yeast
23
or PRT1 of
Arabidopsis
24
(A. Bachmair, unpublished).
In addition, two types of multisubunit RING
enzymes have been described that are conserved
among fungi, animals and plants. One complex type
of RING-finger-containing E3 is called anaphase-
promoting complex (
APC). It has 12 or more subunits
and its function is essential for cell-cycle
progression
25,26
. To date, this type of E3 is poorly
characterized in plants. The second type is called
the SCF complex and has approximately four
subunits
27
. One subunit contains the RING finger
and interacts with an E2. Another contains a so-
called F-box and interacts with substrates; this
subunit usually has additional protein-interaction
domains, such as leucine-rich repeat (LRR)
domains or WD40 repeats
27,28
. Another subunit
contains a cullin-homology domain and serves as a
scaffold with multiple contacts to the other
subunits. One subunit anchors the F-box protein to
the scaffold.
TRENDS in Plant Science
Vol.6 No.10 October 2001
http://plants.trends.com
465
Review
AGI:
Arabidopsis
Genome Initiative, the consortium of scientists
that sequenced the
Arabidopsis
genome
APC:Anaphase promoting complex, a multisubunit ubiquitin
protein ligase (E3) complex
GUS:β-glucuronidase from
E. coli
, a reporter gene frequently
used in plants
I
κκ
B:Inhibitor of NFκB, mammalian protein that keeps NFκB in the
cytoplasm and thereby inhibits NFκB´s function in gene activation
and repression
N-end rule: Ubiquitin-dependent degradation pathway that
recognizes bulky first amino acids of proteins as degradation
signals
NF
κκ
B:Nuclear factor kappa B, a mammalian transcription factor
Orthologs:Genes from different organisms that evolved from a
common ancestor gene and have the same function
Rub:Related to ubiquitin, a ubiquitin-like protein with separate
conjugation system
SCF:Skp1, Cullin, and F-box subunit containing ubiquitin protein
ligase (E3) complex
SUMO:Small ubiquitin-like modifier, a ubiquitin-like protein that
has its own conjugation system
TAIR:The
Arabidopsis
Information Resource
(www.arabidopsis.org)
UBC:Ubiquitin conjugating enzyme
Glossary
Like APC, SCF is involved in the degradation of
key proteins from the cell cycle. However, its range of
substrates extends beyond proteins from the cell
cycle. Database searches for F-box genes suggest that
TRENDS in Plant Science
Vol.6 No.10 October 2001
http://plants.trends.com
466
Review
Gene from
Arabidopsis thaliana
ecotype Columbia
AGI
Name
BAC locus Chr. locus
GenBank
Accession no.
Chr.
Domain
architecture
E2
class
Ubiquitin activating enzymes (E1s)
UBA1
T27E13.15 AT2g30110 AAB39246 2
UBA1
UBA2
MHF15.2 AT5g06460 AAB37569
5
UBC-like with catalytic cysteine (E2s)
UBC1
BAB08733 K3K7.1 AT5g50870 BAB08733 5
UBC1
F14L17.35 AT1g14400 AAA32903
1
UBC2
T20F6.10 AT2g02760 AAC05346
2
UBC2
UBC3
K19B1.15 AT5g62540 AAA32898
5
UBC8
MBK23.24 AT5g41700 AAG40361
5
UBC9
T13J8.70 AT4g27960 AAA32894
4
UBC10/UBCA
K19E1.10 AT5g53300 AAG40069
5
UBC11/UBCB
F17O14.16 AT3g08690 AAG51362
3
BAB09297 MDA7.21 AT5g56150 BAB09297
5
AAD24607 T24I21.15 AT2g16740 AAD24607
2
AAF24583 F22C12.17 AT1g64230 AAF24583
1
AAG51365 F17O14.17 AT3g08700
AAG51365
3
AAF79390 F15O4.1 AT1g35700
AAF79390
1
UBC4
,
UBC5
AAF34303 F7F23.6 AT1g36340 AAG52201
1
BAB09462 MXI22.15 AT5g50430 BAB09462
5
AAD50006 F20D23.1 AT1g17280
AAD50006
1
UBC6
BAA94978 K14A17.12 AT3g17000 BAA94978
3
UBC7
Mnc17.190 AT5g59300 AAC49321
5
UBC13
F18L15.180 AT3g46460 AAC49322
3
UBC7
UBC14
T22E16.40 AT3g55380 AAC49323
3
UBC4
MYC6.5 AT5g41340 AAA32900
5
UBC5
T12P18.18 AT1g63800 P42749
1
UBC8
UBC6
T3F17.32 AT2g46030 AAB32508
2
TF
TF
TF
Ct
Ct
TF TF
Ct
TF TF
Ct
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4
4
4
4
2
2
2
2
2
2
2
2
3
3
UBC10
AT5g25760
F18A17.10
AT5g25760
NA
5
BAB01863 MAL21.6 AT3g20060
BAB01863
3
UBC11
AAG51188 F17J6.3
AT1g50490
AAG51188
1
AT1g78870
F9K20.8 AT1g78870
NA
1
UBC13
AAF99844
F6I1.34
AT1g16890
AAF99844
1
Potential ubiquitin conjugating enzymes with no ortholog in
S. cerevisiae
UBC15/UBC2-1
F27F5.13 AT1g45050 AAC39324
1
UBC18
MBD2.19 AT5g42990 BAB09201 5
UBC16
F1B16.9 AT1g75440 AAG13066 1
AtUBC15
-like
UBC17
C7A10.950 AT4g36410 AAC39326 4
AAF35401 MJK13.1 AT3g15355 AAF35401
3
AAC64223 F12A24.10 AT2g16920 AAC64223 2
NA F8L10.22 AT1g53020 AC022520(NT) 1
hKIAA1734
AAC69130 T1B8.8 AT2g33770 AAC69130 2
hUBCE BAB11530 MUG13.6 AT5g05080 BAB11530 5
Basic tail Acidic tail ThiF
Transmembrane
UBA UBACT UBCC
A
A
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
A
TRENDS in Plant Science
Gene from
Saccharomyces
cerevisiae
3
plants make extensive use of the SCF type of
ubiquitin–protein ligases. Interestingly, the number
of scaffolding (cullin) subunits (ten are known) is
much smaller than the number of substrate-
interacting F-box subunits (>500; Table 1). This
suggests a modular setup of SCF E3 enzymes in
plants. Further analysis of F-box subunits should be
particularly rewarding. The range of mutant
phenotypes encountered to date suggests that each
F-box protein has a specific role in a particular
cellular process, such as in the jasmonic acid
response
29
or the circadian clock
30,31
. Identification of
substrates for these E3 enzymes will certainly help us
to understand how proteolytic events are integrated
into metabolic and regulatory processes.
Ubiquitin chain topology
Although none of the recent progress in
understanding ubiquitin chains was made using
plants as experimental organisms, the results are
almost certainly relevant to plants, given the good
conservation of components involved in
ubiquitylation. Ubiquitin can be linked singly to a
substrate. The linkage is an isopeptide bond
between Gly76 of ubiquitin and an internal Lys of
the substrate. Although some proteins with one
ubiquitin attached, such as histones H2A or H2B,
are metabolically stable, the addition of a single
ubiquitin has been shown to target certain
membrane proteins for degradation in the
lysosome
4
. Alternatively, a few or even many
ubiquitin moieties can be attached to the
substrate
1
. The first ubiquitin linkage is usually
formed with an internal Lys residue of the
substrate. However, some substrates can receive
their first ubiquitin moiety attached to the α-amino
group of the N-terminus
32
. In the well studied cases
of multiubiquitylation, the subsequent ubiquitin
moieties are attached to a previously linked
ubiquitin, not directly to the substrate (Fig. 1).
Recent work has increased our understanding of
the generation and significance of such ubiquitin
‘trees’or chains.
Ubiquitin moieties linked to each other via Gly76
and Lys48 (of ubiquitin) in an isopeptide bond bind to
the proteasome
33
, and this type of multiubiquitin
chain leads to degradation of the substrate by the
proteasome. In line with the significance of
proteasomal protein turnover, Lys48 linkages are
essential in yeast
34
and probably also in plants
35
. The
N-end-rule E3 UBR1 (Ref. 23) has been reported to
produce only this type of linkages, and the same can
be assumed for most E3 enzymes that direct the
degradation of substrate proteins.
By contrast, an abundant intracellular
multiubiquitylated protein, the ribosomal protein
L28, is metabolically stable
36
. This protein carries a
Gly76-to-Lys63-linked multiubiquitin chain. The
complex of ScUBC13 with the E2-like protein
ScMMS2 forms such linkages
20
. ScMMS2 had been
defined genetically as a DNA-repair enzyme. These
findings suggest that part of ubiquitin’s elusive role
in DNA repair involves something other than
marking proteins for degradation. Instead, Lys63
chains might help to recruit additional repair
components or to attract unfolding complexes for
local disassembly of protein–DNA complexes. In
animals, Lys63 chains generated by the homologs of
ScUBC13 and ScMMS2 are involved in the
activation of a protein kinase complex
37
. Lys63
linkages made by the HECT domain E3 ScRSP5
were reported to increase the lysosomal degradation
of a membrane protein that is already marked for
lysosomal degradation by monoubiquitylation
38
.
There is no clear ortholog of the HECT domain E3
ScRSP5 in Arabidopsis; the closest proteins are even
closer to ScTOM1 (Fig. 5).
The third type of ubiquitin chain discussed here
involves Gly76–Lys29 linkages. An E3 enzyme
implicated in generating this type of linkage,
ScUFD4, is of the HECT-domain type
39
(Fig. 5).
The significance of this step is still unclear.
Interestingly, ScUFD4 needs another, post-E3,
factor, the E4 UFD2 (Fig. 5), to elongate a short
ubiquitin chain
40
. The added chain is probably
linked through Lys48 and thus allows proteasomal
degradation of the substrate.
Ubiquitylation substrates
In the plant field, remarkable progress has been
made, and more can be expected in the near future,
regarding the identification of ubiquitylation
substrates. Although oat phytochrome A is still the
only plant protein for which ubiquitylated forms
have been detected in vivo
41
, several recent
publications analyze degradation signals (degrons) of
plant proteins and tentatively assign them to E3
pathways. The Aux/IAA group of transcriptional
regulators is metabolically unstable and their
instability is essential for their function because a
TRENDS in Plant Science
Vol.6 No.10 October 2001
http://plants.trends.com
467
Review
Fig. 3. Genes encoding ubiquitin-activating and putative ubiquitin-
conjugating enzymes (E1s and E2s). The domain designations used are:
ThiF, NAD/FAD binding fold found in E1s and ThiF/MoeB/HesA family
members (PF00899); UBA, ubiquitin-associated domain (PF00627);
UBACT, repeat domain in the C-terminus of E1s (PF02134); UBCC,
catalytic domain of E2s (PF00179). E2s have been divided into four
classes according to Ref. 14. Class 1 members contain only the UBC
domain. Class 2 members have a C-terminal extension, which can be
involved in localization, as in the case of the integral membrane
subfamily UBC6. Class 3 members have an N-terminal extension.
Class 4 consists of the remaining sequences. There are several
sequence inconsistencies with ubiquitin-conjugating-enzyme-type
proteins in the databases. For AT1g64230, the GenBank and especially
the
Arabidopsis
Genome Initiative (AGI) protein entry contain
mispredictions of the protein sequence. In the case of AT1g35700,
there is no protein sequence in the AGI database. AT5g59300 has a
mispredicted N-terminus in the AGI database. The AT1g53020 gene
appears to contain three UBCC domains in the AGI database, a
prediction that cannot be supported by related proteins. Alternatively,
three proteins have been predicted in the same region (AAF87864,
AAG52279, AAG52276). Abbreviations: CHR, chromosome; NA, not
available; NT, nucleotide (sequence). The
Arabidopsis
sequence sets
of the AGI used in this work were obtained from the TAIR website
(http://www.arabidopsis.org/) on March 22nd, 2001.
mutation that interferes with turnover causes an
auxin-insensitive phenotype
42
. The elusive
degradation signal of phytochrome A has been more
narrowly defined
43
. The RING-finger protein COP1
was identified as the key player in degradation of the
transcription factor HY5 (Ref. 44). A recent report
indicates that the movement protein of tobacco
mosaic virus (TMV) is a substrate for
ubiquitylation
45
, and ubiquitylation of TMV coat
protein has also been reported
46
. Ubiquitylation of
viral coat proteins also seems to be widespread in
animal viruses. The significance of this step is not
completely clear but the ubiquityl moiety probably
facilitates viral budding
4
.
As in yeast, fusion proteins play an important
role in analysis of substrate degradation. An
artificial substrate for the N-end rule served to
elucidate the corresponding degradation
pathway
24,47
. Afusion of Arabidopsiscyclin B1;1 to
β-glucuronidase (
GUS) is apparently as unstable as
the unfused cyclin
48,49
, and fusions of plant cyclins to
a chloramphenicol acetyl transferase reporter were
used to study the timing and the signals involved in
cell-cycle-regulated degradation
50
. Likewise, a
diverse set of degrons known from yeast were fused
to luciferase and GUS and tested in Arabidopsis
51
,
with the goal of obtaining reporter proteins with a
predetermined, short half-life. Eventually, protein
interaction assays with substrate binding subunits
of E3s might help us to find more in vivo substrates.
Regulation of ubiquitylation
Ubiquitylation processes are tightly regulated and
most degrons are only generated or recognized if
specific conditions are met. The multiplicity of gene
families for most components of the machinery
suggests that transcriptional regulation is
important in plants. It will be interesting to
determine whether newly induced E3 subunits are
incorporated into existing E3 complexes, perhaps
with the help of assembly factors, or whether they
can interact only with unassembled ‘constitutive’
subunits.
Reversible modification of E3 subunits by
phosphorylation is important for the activity of APC-
type ubiquitin–protein ligases (e.g. the yeast subunits
ScAPC1, ScAPC3, ScAPC8 and ScAPC16 can be
modified by phosphate addition). Modification of the
cullin subunit of SCF-type E3 enzymes by the
ubiquitin-like protein RUB1 has been detected in
animals and in plants
11,12,52,53
. Like phosphorylation,
this modification is reversible and leads to changes in
activity
54
. In plants, this modification is essential for
a response to auxin
53
.
The most prevalent form of regulation that
influences ubiquitylation is a change in the
substrate. The conformational change of
phytochrome A upon red light irradiation is believed
to expose degradation signals. For N-end-rule
TRENDS in Plant Science
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AAF87019 F26F24.19 AT1g23260 AAF87019 1
AAG52343 F5A18.16 AT1g70660 AAG52343
1
CAB43411 F22O6.60 AT3g52560 CAB43411 3
MMS2
AAD21451 F11F19.3 AT2g36060 AAD21451 2
AAG51025 T2E22.129 AT3g12400 AAG51025 3
VPS23
BAB11114 MAC12.18 AT5g13860 BAB11114 5
BAB01762 MRP15.19 AT3g13550
BAB01762
3
Ungrouped
UBCC domain
AAC04484 F24L7.7 AT2g32790
AAC04484
2
Gene from
Arabidopsis
thaliana
ecotype Columbia
AGI
Gene from
Saccharomyces
cerevisiae
Name
BAC locus Chr. locus
GenBank
Accession no.
Chr.
Domain
architecture
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
Uc
TRENDS in Plant Science
Fig. 4. Genes encoding
UBC-domain proteins
without the conserved
active-site Cys (ubiquitin
enzyme variants, UEVs).
The UBCC domain is the
catalytic domain of E2s
(PF00179). Two UEV
proteins remain
unclassified. BAB01762
has orthologs in other
plants that form a group
with some similarity to
animal UbcM2 proteins
but lack the catalytic
cysteine and with a more
glycine-rich N-terminal
extension. For AAC04484,
no ESTs could be found.
Abbreviation: Chr.,
chromosome. The
Arabidopsis
sequence
sets of the
Arabidopsis
Genome Initiative (AGI)
used in this work were
obtained from the TAIR
website (http://www.
arabidopsis.org/) on
March 22nd, 2001.
Table 1. Occurrence of proteins with selected domains
involved in ubiquitylation or de-ubiquitylation
(conservative estimates)
Domains
a
Yeast
b
Arabidopsis thaliana
b
Cullin 4/4/2 10/10/6
F-box 11/11/9 558/523/518
HECT 5/5/5 7/7/7
RING 30/21/39 336/283/364
SKP1 2/2/2 21/21/21
U-box –/–/2 –/–/60
UBP 16/16/– 29/29/–
UCH 1/1/– 3/3/–
a
Cullin, Cullin family domain (PF00888); F-box, F-box domain first
described for cyclin-F (PF00646); HECT, domain h
omolgous to
(human papilloma virus) E
6- associated protein carboxyl terminus
(PF00632); RING, 2 Zn
2+
atoms containing domain of RING finger
proteins (really interesting new gene) (PF00097); SKP1,
homologous to ScSKP1 (PF01466); U-box, domain with a RING
finger-like structure (but without Zn
2+
atoms) first identified in
ScUFD2; UBP, ubiquitin proteases containing a Cys and a His box
(UCH-1 (PF00442) and UCH-2 (PF00443)); UCH, ubiquitin carboxyl-
terminal hydrolases (PF01088).
b
The three numbers are obtained with domain descriptions from
public domain libraries (Pfam/SMART) with the indicated E-value
thresholds: Pfam 0.1/ Pfam 0.01/SMART 0.1. These E-value
thresholds are stringent, therefore additional proteins with
functionally relevant similarity to the indicated domains might exist.
The indicated numbers therefore serve as a lower limit for the true
value. A dash (–) indicates data not available.
substrates, either endonucleolytic cleavage (e.g. by
an ESP1-type protease
55
; Fig. 5) or post-translational
arginylation (by ATE1; Ref. 23; Fig. 5) can lead to
exposure of the degradation signal, which consists of
a bulky first amino acid
23
. A whole class of F-box
subunits of SCF ubiquitin–protein ligases binds to
and thus recognizes phosphorylated epitopes on their
substrates. Phosphorylation as a prerequisite for
ubiquitylation is indeed used as a regulatory step for
many substrates in the cell cycle, and for many other
proteins such as the animal defense regulator
IκBα
(Ref. 56).
An unexpected intricacy of the
NF-κB regulation
network is that the kinase complex involved in
phosphorylating IκB is itself activated by
ubiquitylation
37
. Another newly discovered
regulatory influence on IκB degradation is the
modification of this protein by the ubiquitin-like
molecule
SUMO. SUMO is covalently linked precisely
to those Lys residues that are targets for
ubiquitylation. However, SUMOylation does not lead
to turnover and thus counteracts ubiquitylation in
this case
57
. It remains to be seen whether similar
cases will be discovered in plants.
De-ubiquitylation
S. cerevisiae has more de-ubiquitylating enzymes
than E2s (Ref. 15; Fig. 3, Table 1). A deeper
understanding of the biological function of yeast de-
ubiquitylating enzymes is currently restricted to
selected examples. Two structural types of de-
ubiquitylating enzymes can be distinguished: the
ubiquitin C-terminal hydrolase (UCH) family and
the ubiquitin protease (UBP) family
15,58,59
. For both
types, representative protein signatures can be
identified in Arabidopsis(Table 1). The Arabidopsis
UBP family is being investigated genetically and
biochemically
60,61
. Increased sensitivity to the amino
acid analog
L-canavanine has been reported for
knockout mutants
61
.
Known roles for these enzymes include the
recycling of ubiquitin from substrates targeted for
degradation and the processing of ubiquitin
precursors. Both of these activities provide free
ubiquitin for further cycles of ubiquitylation.
De-ubiquitylation also has an as-yet poorly
understood regulatory role. For instance, a
de-ubiquitylating enzyme has been implicated in the
control of mammalian cell growth
59
. Furthermore,
removal of ubiquitin from monoubiquitylated
histones is apparently a prerequisite for chromatin
condensation in mitosis
62
. It is tempting to expect a
similar regulated antagonism between the addition
and removal of ubiquitin for other proteins.
Examples are emerging from animals but none has
yet been described from plants.
Summary
The proportion of the genome that is devoted to
ubiquitylation is larger in plants than in animals or
fungi. Cross-species comparisons show good
conservation of genes encoding ubiquitin, E1 and
E2. Plant proteins with suspected ubiquitin ligase
(E3) function are apparently more diverged, and
orthologs can be identified only in a minority of
cases. Progress in understanding ubiquitylation in
plants will certainly continue to profit from results
obtained with animals or fungi. However, there is
an undiminished need for in planta
experimentation and the results obtained to date
suggest that further exciting connections between
ubiquitylation and other fields of plant biology will
continue to surface.
TRENDS in Plant Science
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469
Review
CAB86042 F9G14.190 AT5g02880 CAB86042 5
UFD4/YKL010C
CAB37516 F20M13.160 AT4g38600 CAB37516 4
UPL1 F14J16.14 AT1g55860 AAF36454 1
TOM1
UPL2 F17O7.23 AT1g70320 AAF36455 1
UFD2/YDL190C
CAC01739 T20K14.10 AT5g15400 CAC01739 5
AAG50969 F11B9.118 AT3g11240 AAG50969 3NA
ATE1/YGL017W
ATE1 mJJ3.10 AT5g05700 Q9ZT48 5NA
ESP1/YGR098C
CAA19812 F7H19.150 AT4g22970 CAA19812 4
ESP HECT UBA U-box
A
A
Gene from
Arabidopsis thaliana
ecotype Columbia
TAIR
Gene from
Saccharomyces
cerevisiae
Name
BAC locus Chr. locus
GenBank
Accession no.
Chr.
Domain
architecture
TRENDS in Plant Science
A
Fig. 5. Selected examples of conserved proteins of ubiquitylation
pathways. The domain designations used are: ESP, protease domain in
ScESP1 homologs; U-box, domain with structural similarity to RING
finger (SMART), as found in ScUFD2; UBA, ubiquitin-associated
domain (PF00627). Abbreviation: Chr., chromosome.
Acknowledgements
M.N. and F.E. are
supported by Boehringer
Ingelheim. A.B.’s work is
supported by the Austrian
Science Foundation FWF
(grant P 13927). We thank
Dan Finley (Harvard
Medical School, Boston,
MA USA) for comments
on the manuscript and
Jan Peters (Research
Institute of Molecular
Pathology, Vienna,
Austria) for advice on APC
modification. Because of
space constraints, we
could not quote all the
publications that we
would have liked to, and
we apologize to
colleagues who
contributed to the field
but were not adequately
referenced in this article.
References
1 Hershko, A. and Ciechanover, A. (1998) The
ubiquitin system. Annu. Rev. Biochem. 67,
425–479
2 Callis, J. and Vierstra, R. (2000) Protein
degradation in signaling. Curr. Opin. Plant Biol.
3, 381–386
3 Estelle, M. (2001) Proteases and cellular regulation
in plants. Curr. Opin. Plant Biol. 4, 254–260
4 Hicke, L. (2001) Protein regulation by
monoubiquitin. Nat. Rev. Mol. Cell Biol. 2, 195–201
5 Ingvardsen, C. and Veierskov, B. (2001)
Ubiquitin- and proteasome-dependent proteolysis
in plants. Physiol. Plant. 112, 451–459
6 Weissman, A.M. (2001) Themes and variations on
ubiquitylation. Nat. Rev. Mol. Cell Biol. 2,
169–178
7 Callis, J. et al. (1995) Structure and evolution of
genes encoding polyubiquitin and ubiquitin-like
proteins in Arabidopsis thaliana ecotype
Columbia. Genetics139, 921–939
8 Rao-Naik, C. et al. (1998) The Rub family of
ubiquitin-like proteins – crystal structure of
ArabidopsisRub1 and expression of multiple
Rubs in Arabidopsis. J. Biol. Chem. 273,
34976–34982
9 Sun, C-W. and Callis, J. (1993) Recent stable
insertion of mitochondrial DNA into an
Arabidopsispolyubiquitin gene by
nonhomologous recombination. Plant Cell 5,
97–107
10 Sun, C-W. et al. (1997) A model for the evolution of
polyubquitin genes from the study of Arabidopsis
thalianaecotypes. Plant Mol. Biol. 34, 745–758
11 Hochstrasser, M. (2000) Evolution and function of
ubiquitin-like protein-conjugation systems. Nat.
Cell Biol. 2, E153–E157
12 Vierstra, R.D. and Callis, J. (1999) Peptide tags,
ubiquitous modifiers for plant protein regulation.
Plant Mol. Biol. 41, 435–442
13 Hatfield, P.M. et al. (1997) The ubiquitin-
activating enzyme (E1) gene family in
Arabidopsis thaliana. Plant J. 11, 213–226
14 Jentsch, S. (1992) The ubiquitin-conjugation
system. Annu. Rev. Genet. 26, 179–207
15 Hochstrasser, M. (1996) Ubiquitin-dependent
protein degradation. Annu. Rev. Genet. 30,
405–439
16 Haas, A.L. and Siepmann, T.J. (1997) Pathways of
ubiquitin conjugation. FASEB J. 11, 1257–1268
17 Hauser, H-P. et al. (1998) A giant ubiquitin-
conjugating enzyme related to IAP apoptosis
inhibitors. J. Cell Biol. 141, 1415–1422
18 Liu, Z. et al. (1992) cDNA cloning of a novel
human ubiquitin carrier protein. J. Biol. Chem.
267, 15829–15835
19 Thoma, S. et al. (1996) Members of two gene
families encoding ubiquitin-conjugating enzymes,
AtUBC1-3 and AtUBC4-6, from Arabidopsis
thalianaare differentially expressed. Plant Mol.
Biol.31, 493–505
20 Hofmann, R.M. and Pickart, C.M. (1999)
Noncanonical MMS2-encoded ubiquitin-
conjugating enzyme functions in assembly of
novel polyubiquitin chains for DNA repair. Cell
96, 645–653
21 Katzmann, D.J. et al. (2001) Ubiquitin-dependent
sorting into the multivesicular body pathway
requires the function of a conserved endosomal
protein sorting complex, ESCRT-I. Cell106,
145–155
22 Azevedo, C. et al. (2001) The U-box protein family
in plants. Trends Plant Sci. 6, 354–358
23 Varshavsky, A. (1996) The N-end rule: functions,
mysteries, uses. Proc. Natl. Acad. Sci. U. S. A.
93,12142–12149
24 Potuschak, T. et al. (1998) PRT1of Arabidopsis
thalianaencodes a component of the plant N-end
rule pathway. Proc. Natl. Acad. Sci. U. S. A.
95,7904–7908
25 Zachariae, W. et al. (1998) Mass spectrometric
analysis of the anaphase promoting complex from
yeast: identification of a subunit related to cullins.
Science279, 1216–1219
26 Zachariae, W. and Nasmyth, K. (1999) Whose end
is destruction: cell division and the anaphase-
promoting complex. Genes Dev. 13, 2039–2058
27 Deshaies, R.J. (1999) SCF and cullin/RING
H2-based ubiquitin ligases. Annu. Rev. Cell. Dev.
Biol.15, 435–467
28 Xiao, W. and Jang, J-C. (2000) F-box proteins in
Arabidopsis. Trends Plant Sci. 5, 454–457
29 Xie, D-X. et al. (1998) COI1: an Arabidopsisgene
required for jasmonate-regulated defense and
fertility. Science280, 1091–1094
30 Somers, D.E. et al. (2000) Zeitlupeencodes a novel
clock-associated PAS protein from Arabidopsis.
Cell101, 319–329
31 Nelson, D.C. et al. (2000) FKF1, a clock-controlled
gene that regulates the transition to flowering in
Arabidopsis. Cell101, 331–340
32 Breitschopf, K. et al. (1998) A novel site for
ubiquitination: the N-terminal residue, and not
internal lysines of MyoD, is essential for
conjugation and degradation of the protein.
EMBO J. 17, 5964–5973
33 Thrower, J.S. et al. (2000) Recognition of the
polyubiquitin proteolytic signal. EMBO J. 19,
94–102
34 Finley, D. et al. (1994) Inhibition of proteolysis
and cell cycle progression in a
multiubiquitination-deficient yeast mutant. Mol.
Cell Biol. 14, 5501–5509
35 Bachmair, A. et al. (1990) Perturbation of the
ubiquitin system causes leaf curling, vascular
tissue alterations and necrotic lesions in a higher
plant. EMBO J. 9, 4543–4549
36 Spence, J. et al. (2000) Cell cycle-regulated
modification of the ribosome by a variant
multiubiquitin chain. Cell102, 67–76
37 Deng, L. et al. (2000) Activation of the IκB kinase
complex by TRAF6 requires a dimeric ubiquitin-
conjugating enzyme complex and a unique
polyubiquitin chain. Cell103, 351–361
38 Galan, J-M. and Haguenauer-Tsapis, R. (1997)
Ubiquitin Lys63 is involved in ubiquitination of a
yeast plasma membrane protein. EMBO J. 16,
5847–5854
39 Johnson, E.S. et al. (1995) A proteolytic pathway
that recognizes ubiquitin as a degradation signal.
J. Biol. Chem. 270, 17442–17456
40 Koegl, M. et al. (1999) Anovel ubiquitination
factor, E4, is involved in multiubiquitin chain
assembly. Cell96, 635–644
41 Shanklin, J. et al. (1987) Red light-induced
formation of ubiquitin–phytochrome conjugates:
identification of possible intermediates of
phytochrome degradation. Proc. Natl. Acad. Sci.
U. S. A. 84, 359–363
42 Worley, C.K. et al. (2000) Degradation of Aux/IAA
proteins is essential for normal auxin signalling.
Plant J. 21, 553–562
43 Clough, R.C. et al. (1999) Sequences within both
the N- and C-terminal domains of phytochrome A
are required for PFR ubiquitination and
degradation. Plant J. 17, 155–167
44 Osterlund, M.T. et al. (2000) Targeted
destabilization of HY5 during light-regulated
development of Arabidopsis. Nature405, 462–466
45 Reichel, C. and Beachy, R.N. (2000) Degradation
of tobacco mosaic virus movement protein by the
26S proteasome. J. Virol. 74, 3330–3337
46 Hazelwood, D. and Zaitlin, M. (1990)
Ubiquitinated conjugates are found in
preparations of several plant viruses. Virology
177, 352–356
47 Bachmair, A. et al. (1993) Use of a reporter
transgene to generate Arabidopsismutants in
ubiquitin-dependent protein degradation. Proc.
Natl. Acad. Sci. U. S. A. 90, 418–421
48 Colón-Carmona, A. et al. (1999) Spatio-temporal
analysis of mitotic activity with a labile
cyclin–GUS fusion protein. Plant J. 20, 503–508
49 Criqui, M.C. et al. (2000) Cell cycle-dependent
proteolysis and ectopic overexpression of cyclin B1
in tobacco BY2 cells. Plant J. 24, 763–773
50 Genschik, P. et al. (1998) Cell cycle-dependent
proteolysis in plants: identification of the
destruction box pathway and metaphase arrest
produced by the proteasome inhibitor MG132.
Plant Cell 10, 2063–2075
51 Worley, C.K. et al. (1998) Engineering in vivo
instability of firefly luciferase and Escherichia coli
β-glucuronidase in higher plants using
recognition elements from the ubiquitin pathway.
Plant Mol. Biol. 37, 337–347
52 del Pozo, J.C. and Estelle, M. (2000) F-box
proteins and protein degradation: an emerging
theme in cellular regulation. Plant Mol. Biol.
44,123–128
53 Gray, W.M. and Estelle, M. (2000) Function of the
ubiquitin–proteasome pathway in auxin
response. Trends Biochem. Sci. 25, 133–138
54 Schwechheimer, C. et al. (2001) Interactions of the
COP9 signalosome with the E3 ubiquitin ligase
SCF
TIR1
in mediating auxin response. Science
292,1379–1382
55 Rao, H. et al. (2001) Degradation of a cohesin
subunit by the N-end rule pathway is essential for
chromosome stability. Nature410, 955–959
56 Karin, M. and Ben-Neriah, Y. (2000)
Phosphorylation meets ubiquitination: the control
of NF-κB activity. Annu. Rev. Immunol. 18,
621–663
57 Müller, S. et al. (2001) Sumo, ubiquitin’s
mysterious cousin. Nat. Rev. Mol. Cell Biol. 2,
202–210
58 Chung, C.H. and Baek, S.H. (1999)
Deubiquitinating enzymes: their diversity and
emerging roles. Biochem. Biophys. Res. Commun.
266, 633–640
59 D’Andrea, A. and Pellman, D. (1998)
Deubiquitinating enzymes: a new class of
biological regulators. Crit. Rev. Biochem. Mol.
Biol. 33, 337–352
60 Rao-Naik, C. et al. (2000) Ubiquitin-specific
proteases from Arabidopsis thaliana: cloning of
AtUBP5and analysis of substrate specificity of
AtUBP3, AtUBP4, and AtUBP5 using
Escherichia coli in vivo and in vitro assays. Arch.
Biochem. Biophys. 379, 198–208
61 Yan, N. et al. (2000) The ubiquitin-specific
protease family from Arabidopsis. AtUBP1 and 2
are required for the resistance to the amino acid
analog canavanine. Plant Physiol. 124,
1828–1843
62 Hirano, T. (2000) Chromosome cohesion,
condensation, and separation. Annu. Rev.
Biochem. 69, 115–144
TRENDS in Plant Science
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Review
