The EMBO Journal Vol.17 No.8 pp.2208–2214, 1998
A novel protein modification pathway related to the
Dimitris Liakopoulos, Georg Doenges,
Kai Matuschewski and Stefan Jentsch1
ZMBH, Zentrum fu ¨r Molekulare Biologie der Universita ¨t Heidelberg,
Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany
Ubiquitin conjugation is known to target protein sub-
strates primarily to degradation by the proteasome or
via the endocytic route. Here we describe a novel
protein modification pathway in yeast which mediates
the conjugation of RUB1, a ubiquitin-like protein
displaying 53% amino acid identity to ubiquitin. We
show that RUB1 conjugation requires at least three
proteins in vivo. ULA1 and UBA3 are related to
the N- and C-terminal domains of the E1 ubiquitin-
activating enzyme, respectively, and together fulfil E1-
like functions for RUB1 activation. RUB1 conjugation
also requires UBC12, a protein related to E2 ubiquitin-
conjugating enzymes, which functions analogously to
E2 enzymes in RUB1–protein conjugate formation.
Conjugation of RUB1 is not essential for normal cell
growth and appears to be selective for a small set
of substrates. Remarkably, CDC53/cullin, a common
subunit of the multifunctional SCF ubiquitin ligase,
was found to be a major substrate for RUB1 con-
jugation. This suggests that the RUB1 conjugation
pathway is functionally affiliated to the ubiquitin–
proteasome system and may play a regulatory role.
Ubiquitin is an abundant, highly conserved 76 amino
acid protein found in all eukaryotic cells either free or
covalently attached to cellular proteins (reviewed by
Hochstrasser, 1996; Varshavsky, 1997). Conjugation of
ubiquitin to other proteins involves the formation of an
isopeptide bond between the C-terminal glycine residue
of ubiquitin and the ε-amino group of a lysine residue of
an acceptor protein. All known functions of ubiquitin are
thought to be mediated through this reaction. Conjugated
ubiquitin can be a substrate for further ubiquitination
reactions and, indeed, most substrates appear to be modi-
fied by multiubiquitin chains in which single ubiquitin
molecules are linked via isopeptide bonds. Depending on
of another ubiquitin molecule, different types of multi-
ubiquitin chains can be formed in vivo (Hochstrasser,
conjugates appear to be targeted to the 26S proteasome,
© Oxford University Press
which degrades the substrates to small peptides, whereas
ubiquitin is recycled. Certain cell surface proteins, how-
ever, when they are modified by ubiquitination, appear to
be targeted for lysosomal degradation via the endocytic
route (Ko ¨lling and Hollenberg, 1994; Galan et al., 1996;
Hicke and Riezman, 1996; Galan and Haguenauer-Tsapis,
1997). It can therefore be assumed that ubiquitin functions
primarily as a post-translationally added targeting module
directing the conjugated substrates to different proteo-
Conjugation of ubiquitin proceeds via a reaction cascade
involving ubiquitin-activating (E1), ubiquitin-conjugating
(E2) enzymes and, at least in some cases, ubiquitin–
protein ligases (E3) (Ciechanover, 1994; Jentsch and
Schlenker, 1995; Scheffner et al., 1995; Hochstrasser,
1996; Varshavsky, 1997). The E1 enzyme hydrolyses ATP
and, via an E1-bound ubiquitinyl adenylate intermediate,
forms a high energy thioester between a cysteine of its
active site and the C-terminus of ubiquitin. Ubiquitin is
then passed on to E2 enzymes, which form thioester-
linked complexes with ubiquitin in a similar fashion.
Finally, ubiquitin is covalently attached to the substrate
protein by the E2 enzymes or, alternatively, by E3 enzymes
which may possess substrate-binding properties (Scheffner
et al., 1995). In yeast, E1 is encoded by a single gene,
UBA1 (McGrath et al., 1991), whereas families of E2 and
E3 enzymes exist, indicating that E2 and E3 enzymes
mediate the specificity of the system (Jentsch, 1992;
Hochstrasser, 1996; Varshavsky, 1997)
The recent discoveries of ubiquitin-like proteins in
apparently all eukaryotic cells suggest that post-transla-
tional modification of proteins by the covalent attachment
of other proteins is more common than previously
expected. In contrast to ubiquitin, these proteins seem to
play non-proteolytic roles. Conjugation of the mammalian
protein UCRP, a distant ubiquitin relative resembling two
tandem copies of ubiquitin, appears to target conjugates
to the cytoskeleton (Haas et al., 1987; Loeb and Haas,
1992, 1994). SUMO-1, a small ubiquitin-like protein from
higher eukaryotic cells, was found covalently linked to
RanGAP1, the activating protein of the Ran GTPase
involved in the regulation of nucleocytoplasmic trafficking
(Matunis et al., 1996; Johnson and Hochstrasser, 1997;
Mahajan et al., 1997; Saitoh et al., 1997). Conjugation of
SUMO-1 to RanGAP1 targets the otherwise cytosolic
protein to the nuclear pore complex (Matunis et al., 1996;
Mahajan et al., 1997). SMT3, a yeast ubiquitin-like protein
displaying 50% sequence identity to SUMO-1, is essential
for viability, but its cellular function is presently unknown
(Johnson et al., 1997). The enzymes involved in the
conjugation of SMT3 to other proteins have been identified
recently. Two proteins, AOS1 and UBA2, are needed for
SMT3 activation (Dohmen et al., 1995; Johnson et al.,
1997). Interestingly, AOS1 resembles the N-terminal
Novel protein modification pathway in yeast
Fig. 1. Sequence comparison of RUB1 (DDBJ/EMBL/GenBank
accession No. Y16890) with mouse NEDD8, yeast SMT3, and
ubiquitin. Residues which are conserved between ubiquitin and the
ubiquitin-like proteins are boxed. Processing sites of the precursors are
indicated by an arrow.
domain, whereas UBA2 resembles the C-terminal domain
of the E1 ubiquitin-activating enzyme, UBA1. This indi-
cates that the two proteins form a heterodimer with E1-
like activity (Johnson et al., 1997). Conjugation of SMT3
and SUMO-1 also involves an E2 enzyme, UBC9, which
closely resembles ubiquitin-conjugating enzymes structur-
ally (Johnson and Blobel, 1997; Saitoh et al., 1998;
Schwarz et al., 1998).
In this study, we identify a novel ubiquitin-like protein
from yeast, RUB1, which appears to be conserved from
yeast to mammals. Similarly to ubiquitin, RUB1 can be
conjugated to other proteins in vivo. We describe the
mechanism by which RUB1 is conjugated and identify
the enzymes required for RUB1 activation and conjugation
in vivo. Intriguingly, CDC53/cullin, a common subunit of
the SCF ubiquitin ligase, was found to be a major substrate
of this novel post-translational modification system,
indicating that RUB1 conjugation may regulate ubiquitin
RUB1, a novel ubiquitin-like protein from yeast
In a search for new components of ubiquitin-related
pathways, we identified in the yeast Saccharomyces cere-
visiae genome a gene for a novel ubiquitin-like protein
termed RUB1 (related to ubiquitin 1; Hochstrasser, 1996;
Figure 1). The RUB1 open reading frame (ORF) is
interrupted by a single intron (at codon 50) and encodes
a 77 residue protein which is particularly related (59%
identity) to the mammalian ubiquitin-like protein NEDD8
(Kumar et al., 1992), suggesting that the two proteins are
functional orthologues. RUB1 displays 53% sequence
identity with ubiquitin and 18 and 25% with the ubiquitin-
like proteins SMT3 from yeast (Johnson et al., 1997) and
SUMO-1 from higher eukaryotes (Matunis et al., 1996;
Mahajan et al., 1997), respectively. Interestingly, most
residues conserved between RUB1 and ubiquitin cluster
on one side of the three-dimensional structure of ubiquitin
and, in particular, to a domain close to the C-terminus
through which ubiquitin is conjugated to other proteins
(Figure 2). This conservation led us to speculate that
RUB1 may be conjugated to other proteins analogously
to ubiquitin (see below).
To assess the in vivo function of RUB1, yeast strains
Fig. 2. Residues conserved between ubiquitin and RUB1 drawn into
the known structure of ubiquitin (Vijay-Kumar et al., 1987). Identical
residues are shown in white, similar residues in light green, and
dissimilar residues in dark green. Left and right views are shown, and
the C-terminus of ubiquitin through which ubiquitin is attached to
other proteins is indicated. Note that identical residues are clustered on
the right side of the molecule and at a domain close to the C-terminus.
The coordinates were obtained from the Brookhaven database and
displayed by Insight™ software (Biosym Technology).
lacking the RUB1 gene were constructed. Conventional
gene replacements, however, affected the expression of
an adjacent gene (not shown). We therefore generated a
null mutant by introducing a frameshift at codon 15 of
the RUB1 sequence. The introduction of the frameshift
allele into the genome couldbe followed by the appearance
yeast mutants lacking functional copies of RUB1 were
viable and showed no detectable mutant phenotype (not
shown). The null mutants grew at wild-type rates and
displayed no obvious defects when grown under starvation
or other stress conditions (e.g. heat stress, cadmium,
canavanine). Thus, unlike ubiquitin (O ¨zkaynak et al.,
1987; Varshavsky, 1997) or SMT3 (Johnson et al., 1997),
RUB1 is not essential for normal cell growth and viability.
RUB1 is conjugated to other proteins in vivo
The predicted primary translation product of RUB1 con-
tains 77 amino acid residues and terminates C-terminally
with two glycine residues followed by a single asparagine
proteolytic cleavage C-terminal of a similarly positioned
double glycine sequence, and becomes conjugated to other
proteins via the processed C-terminus (O ¨zkaynak et al.,
1987; Varshavsky, 1997), we postulated that RUB1 might
be subject to an analogous fate. To identify possible RUB1
conjugation reactions in vivo, we constructed an epitope-
tagged variant of RUB1 by fusing sequences encoding a
haemagglutinin (HA) epitope to the N-terminus of RUB1
(HARUB1). When this construct was expressed in wild-
type yeast cells, Western blot analysis detected a protein
of the predicted size ofHARUB1 (Figure 3). Notably, a
limited number of additional HA-antibody-reactive pro-
teins with sizes ranging from 30 to ~100 kDa could be
identified, suggesting that these proteins represent RUB1–
protein conjugates formed in vivo (Figure 3). Interestingly,
these additional proteins could also be detected when we
used a variant ofHARUB1 lacking the C-terminal aspara-
gine residue, indicating that, analogously to ubiquitin,
RUB1 matures to a protein with a C-terminal double
glycine motif. The extra immunoreactive proteins were
D.Liakopoulos et al.
absent when anHARUB1 derivative was expressed which
lacked the C-terminal glycine residues (Figure 3). We thus
conclude that after proteolytic removal of the C-terminal
asparagine residue from RUB1’s precursor, the matured
76 residue RUB1 protein can be conjugated to a limited
number of other proteins via its C-terminal glycine residue.
RUB1 activation is mediated by the ULA1–UBA3
E1 enzyme pair
The observation that RUB1 can be attached to other
proteins in vivo suggested that this reaction may also
involve specific activating and conjugating enzymes.
Activation of ubiquitin is mediated by an ~100 kDa E1
enzyme, and the yeast enzyme, UBA1, is encoded by a
gene essential for viability (McGrath et al., 1991). In
Fig. 3. Conjugate formation of RUB1 and RUB1 variants. Expression
of N-terminally HA-tagged RUB1 (HARUB1) in wild-type yeast leads
to the formation of a set ofHARUB1–protein conjugates (detection by
Western blot).HARUB1 without the C-terminal Asn residue (GG) is
conjugated, butHARUB1 lacking the C-terminal sequence GlyGlyAsn
(∆GGN) is not.
Fig. 4. (A) Protein sequences of ULA1, UBA3 and UBC12 (DDBJ/EMBL/GenBank accession Nos. Y16889, Y16891 and X9942, respectively). The
putative active site cysteines in UBA3 and UBC12 are marked by an asterisk. (B) Schematic representation of the similarity domains between UBA1
and related proteins. The putative active site cysteines are located within similarity box III (termed the UBA domain), as indicated. The similarity
boxes correspond to those of Johnson et al. (1997).
UBA1-related genes. Two of them, AOS1 and UBA2,
were shown recently to encode proteins which form a
heterodimer with activating activity for the ubiquitin-like
protein SMT3 (Dohmen et al., 1995; Johnson et al., 1997).
Since no difference in RUB1 conjugation was found in
either uba1 or uba2 temperature-sensitive mutants (not
shown), we postulated that other enzymes might be needed
for RUB1 activation.
Indeed,we identifiedtwoadditional UBA1-relatedgenes
in the yeast genome which we termed ULA1 (ubiquitin-
like protein activation) and UBA3 (ubiquitin-like protein-
activating enzyme). ULA1 encodes a protein (52.9 kDa;
462 amino acids; Figure 4A) which displays significant
similarities to the N-terminal domain of UBA1 and, in
particular, resembles AOS1 structurally over its entire
length (Figure 4B). The other gene, UBA3, encodes a
smaller protein (33.3 kDa; 299 amino acids; Figure 4A)
which is homologous to UBA2 and to the C-terminal
domain of UBA1 (Figure 3B). Notably, the UBA3 protein
bears a cysteine residue at a position similar to the
active site cysteine residues of UBA1 and UBA2 (within
similarity box III; Figure 4) required for ubiquitin and
SMT3 activation, respectively. Given these similarities,
we considered the possibility that, analogous to the AOS1–
UBA2 pair, ULA1 and UBA3 may combine to form an
enzyme with E1-like activity for RUB1 activation, with
UBA3 being the catalytically active component of the
complex. To test these ideas, we generated null mutants
of both genes. The ULA1 gene was completely replaced
by the yeast LEU2 gene and a uba3 null mutant was
generated by replacing 60% of the UBA3 ORF by the
HIS3 marker gene. The phenotypes of the null mutants
subsequently were analysed after tetrad dissection of
diploid heterozygous mutants. Similar to strains lacking
RUB1, ula1 and uba3 knock-out strains are viable and
exhibit no obvious growth defects (not shown).
Using these mutants, we next tested whether ULA1 and
Novel protein modification pathway in yeast
Fig. 5. The RUB1 conjugation pathway. (A) In vivo conjugation of
HARUB1 in ula1, uba3 and ubc12 null mutants is not detected,
compared with wild-type. (B) Thioester complex formation between
33P-labelled RUB1 and the enzymes of the RUB1 conjugation system.
In addition to [33P]RUB1 and ATP, assays contain control extracts of
insect and bacterial cells (lane 1), and GST–ULA1, UBA3 and
UBC12, as indicated (lanes 2–6). The bands represent thioester-linked
complexes of UBA3 with RUB1 (UBA3–RUB1) and of UBC12 with
RUB1 (UBC12–RUB1). Thioester formation requires the presence of
ULA1. Thioester-linked complexes disappear after boiling under
reducing conditions (lanes 7 and 8).
UBA3 are required for RUB1–protein conjugate formation
in vivo. Indeed, when we expressedHARUB1 in each of
the two mutants, onlyHARUB1 but no additional HA-
antibody-reactive proteins could be identified (Figure 5A).
We thus conclude thatHARUB1 conjugation to cellular
yeast proteins depended on the presence of both ULA1
and UBA3. Furthermore, we asked whether ULA1–UBA3
functions enzymatically similarly to the E1 ubiquitin-
activating enzyme by assaying for thioester formation of
the recombinant proteins with radiolabelled RUB1 in the
presence of ATP in vitro. For these assays, ULA1 was
expressed as a fusion with glutathione-S-transferase (GST)
and purified from Escherichia coli cells, whereas UBA3
was expressed by the baculovirus expression system in
insect cells. As shown in Figure 5B, only protein mixtures
containing both ULA1 and UBA3 mediated the formation
of a radiolabelled complex of ~40 kDa, consistent with
the size of an adduct between RUB1 and UBA3 (Figure
5B, lane 5). This complex was sensitive to boiling under
reducing conditions, indicating that the proteins are indeed
linked via a thioester bond (Figure 5B, lane 7). Neither
complex with RUB1. This indicates that a ULA1–UBA3
pair is needed for RUB1 activation, both in vitro and
RUB1 conjugation is mediated by the UBC12 E2
Yeast cells possess 13 different E2 enzymes which bear
a conserved UBC domain in which the active site cysteine
residue is located (Jentsch et al., 1990; Jentsch, 1992).
One of these enzymes, UBC9, was shown recently to be
specific for the ubiquitin-like protein SMT3 (Johnson and
Blobel, 1997; Saitoh et al., 1998; Schwarz et al., 1998),
but most other E2s are known to mediate ubiquitin
conjugation. We investigated whether any of these E2s
may function in the RUB1 conjugation pathway. When
we expressedHARUB1 in all 13 different mutant strains,
no difference in RUB1 conjugation compared with wild-
type was found (not shown), with the exception of ubc12
null mutants (Figure 5A). In these strains, virtually no
RUB1–protein conjugates were detected. UBC12 (21.2
kDa; 188 amino acids; Figure 4A) carries the hallmark of
a typical E2, i.e. a UBC domain containing a putative
active site cysteine residue for thioester formation at a
conserved position. Similar to other genes of the RUB1
system, UBC12 is not essential for viability (not shown).
When we added an extract from E.coli cells expressing
UBC12 to our thioester assays (see above), we detected,
in addition to the UBA3–RUB1 thioester, a band of
~30 kDa, consistent with the size of a UBC12–RUB1
thioester complex (Figure 5B, lane 6). Complex formation
was dependent on ULA1 and UBA3 in the assay and,
characteristically for a thioester complex, the adduct was
sensitive to boiling under reducing conditions (Figure 5B,
to ubiquitin-conjugating enzymes, but is part of the RUB1
CDC53/cullin is a major substrate of the RUB1
Although the pathway of RUB1 conjugation is remarkably
similar to that of the ubiquitin system, the small number
of detectable RUB1–protein conjugates (see Figure 3) is
strikingly different from the numerous ubiquitin–protein
conjugates present in yeast cells in vivo (Seufert and
Jentsch, 1990). This suggests that RUB1 conjugation is
highly selective and probably not involved in bulk protein
degradation. To investigate the cellular role of RUB1
conjugation, we decided to identify the major substrates
of this pathway. One prominent
(~100 kDa) was particularly abundant in nuclear fractions
(not shown). We isolated this conjugate from anHARUB1-
expressing yeast culture by a procedure involving anti-
HA affinity chromatography (see Materials and methods;
Figure 6A). The eluted material was purified further by
SDS–gel electrophoresis. The ~100 kDa protein was
excised from the gel and tryptic fragments of the protein
were analysed by mass spectrometry. The determined
sizes of 17 fragments corresponded to predicted tryptic
fragments of CDC53, a 94 kDa yeast protein required for
G1–S cell cycle progression (Mathias et al., 1996; Willems
et al., 1996). Intriguingly, CDC53 (also known as yeast
cullin) is a common subunit of the SCF ubiquitin ligase,
an enzyme complex which catalyses the conjugation of
ubiquitin to specific cellular substrates which are then
degraded by the proteasome (see Discussion). This sug-
gests that the RUB1 conjugation system is functionally
tied to the ubiquitin–proteasome-dependent proteolytic
system. To confirm the identity of the conjugate, we
expressed epitope-tagged CDC53 (HXCDC53) in wild-
type and various null mutants of the RUB1 pathway.
HXCDC53 of wild-type cells was found to run as a
doublet in SDS gels, but the slower migrating protein was
completely absent in rub1, ula1, uba3 and ubc12 null
mutants (Figure 6B, and data not shown). When we
expressed a larger variant of RUB1 (HARUB1) in the rub1
null mutant, an even slower migrating antibody-reactive
band appeared. Modification ofHXCDC53 byHARUB1
was verified further by immunoprecipitation and Western
D.Liakopoulos et al.
Fig. 6. Identification of CDC53 as an in vivo substrate of the RUB1
system. (A) Silver-stained gel showing an ~100 kDa conjugate purified
by anti-HA–Sepharose. The protein (indicated by an arrowhead) was
only present in extracts fromHARUB1-expressing cells but not in
control extracts. (B) N-terminally tagged CDC53 (HXCDC53) is
modified in vivo by RUB1 in wild-type cells (WT) but the
modification (protein bands indicated with asterisks) is absent in rub1
and ubc12 null mutants. In rub1 mutants expressingHARUB1, a larger
conjugate withHXCDC53 appears. (C) Immunoprecipitation of
HARUB1 results in co-precipitation of Xpress™-tagged CDC53
(HXCDC53, upper band), which is detectable by tag-specific antibodies
(Invitrogen) in a Western blot (HARUB1). Co-precipitation of
HXCDC53 is not observed in a ubc12 null mutant (ubc12∆). The lower
bands correspond to heavy and light antibody chains.
blotting (Figure 6C). We thus conclude that a fraction of
CDC53 is modified by a single RUB1 moiety in a reaction
which requires the UBC12 E2 enzyme. This modification
was resistant to reducing agents (not shown; Willems
et al., 1996), indicating that RUB1 is not linked via a
thioester, but most likely via an isopeptide bond analogous
to ubiquitin linkages. We suggest that the previous report
ation was possibly mistaken as anti-ubiquitin antibodies
had been employed which readily react with RUB1 (not
A novel ubiquitin-related protein
Ubiquitin is one of the most highly conserved eukaryotic
proteins known to date. Human ubiquitin differs from its
yeast homologue by only three amino acid residues (out
of 76) and the proteins are functionally equivalent. In
addition to ubiquitin, eukaryotes also express ubiquitin-
related proteins, and some of these proteins can be found
with conserved sequences in animals, plants and yeasts.
Two classes of ubiquitin-related proteins can be distin-
guished. Proteins of the first class lack the double glycine
motif at the C-terminal end of the ubiquitin moiety which
is required for precursor processing and conjugation.
Consequently, these proteins are not conjugated to other
cellular proteins. The ubiquitin-related proteins of this
class do not seem to promote selective proteolysis but
play strikingly diverse cellular roles. The yeast ubiquitin-
like protein RAD23, for instance, is implicated in DNA
repair and, together with another ubiquitin-related protein,
DSK2, is also involved in spindle pole body duplication
(Watkins et al., 1993; Biggins et al., 1996). The mamma-
lian ubiquitin-related protein BAG-1 (Takayama et al.,
1995), however, functions as a regulatory cofactor of the
Hsc70 chaperone (Ho ¨hfeld and Jentsch, 1997; Takayama
et al., 1997; Zeiner et al., 1997). The significance of the
ubiquitin-like domain of this class of proteins remains
largely enigmatic, but it has been suggested that these
domains may prime the proteins for ubiquitin-dependent
degradation (Varshavsky, 1997).
Proteins of the second class of ubiquitin-like proteins
are distinguished by their property of becoming post-
translationally attached to other cellular proteins. Known
ently restricted to mammalian cells (Haas et al., 1987;
Loeb and Haas, 1992, 1994), the higher eukaryotic protein
SUMO-1 (Matunis et al., 1996; Johnson and Hochstrasser,
1997; Mahajan et al., 1997; Saitoh et al., 1997; Saitoh
et al., 1998), and SMT3, its apparent yeast orthologue
(Johnson et al., 1997). Also these proteins do not seem
to be involved directly in protein degradation but appear
to function as post-translationally added protein targeting
devices. The yeast protein RUB1, described herein, repre-
sents a novel ubiquitin-like protein of this class. RUB1
exhibits 53% sequence identity with ubiquitin and is
thus the closest homologue of ubiquitin known to date.
Similarly to ubiquitin, RUB1 conjugation in vivo requires
activating and conjugating enzymes. Intriguingly, the
activating enzyme consists of two separate proteins, ULA1
and UBA3, analogous to the AOS1–UBA2 pair required
for SMT3 activation. However, whether the distinct E1
subunits may also assemble to ULA1–UBA2 and AOS1–
UBA3 pairs for other functions is currently speculative.
The E2 enzyme required for RUB1 conjugation in vivo,
UBC12, bears a typical UBC domain and resembles
ubiquitin-conjugating enzymes over its entire length.
Mammalian NEDD8, a likely orthologue of yeast
The RUB1 system described here is most likely evolu-
tionarily conserved. The probable mammalian orthologue
of yeast RUB1 is NEDD8 (Kumar et al., 1992) which
displays 58% sequence identity with RUB1 (Figure 1).
Similarly to RUB1, NEDD8 can be conjugated to other
proteins in vivo (Kamitani et al., 1997). Intriguingly,
expression of NEDD8 in yeast results in a RUB1-like
conjugate pattern that includes NEDD8-modified yeast
CDC53 (D.Liakopoulos and S.Jentsch, in preparation).
Moreover, the size of the major NEDD8 conjugate in
mammalian cells (Kamitani et al., 1997) is consistent with
the predicted size of an NEDD8-modified cullin protein,
suggesting that the NEDD8 pathway is functionally
equivalent to the RUB1 system of yeast. The expression of
NEDD8 is down-regulated during embryonic development
(Kumar et al., 1992). Interestingly, in adult mice, NEDD8
mRNA levels are particularly high in heart and skeletal
muscle (Kamitani et al., 1997). These tissues are character-
ized by a high protein turnover rate and a very limited
capacity to divide. Thus, RUB1/NEDD8 pathways may
be relevant specifically for cells which have exited the
cell cycle (see below).
Novel protein modification pathway in yeast
Cullin as a substrate
Although our current work has not defined the cellular
function of the RUB1 conjugation system, the data pre-
sented indicate that this pathway is affiliated with the
ubiquitin system. Surprisingly, our studies identified
CDC53 as a major substrate of this novel modification
system. CDC53, a member of the evolutionarily conserved
family of cullin proteins (Kipreos et al., 1996), is a
common subunit of the SCF ubiquitin ligase which
ubiquitinates a variety of substrates promoting their
destruction (Mathias et al., 1996; Feldman et al., 1997;
Skowyra et al., 1997). SCF complexes also contain SKP1
and alternative substrate-specific F-box proteins. It has
been shown that the degradation of the cyclin kinase
inhibitor SIC1 is mediated by the UBC3 (CDC34) ubiqui-
tin-conjugating enzyme and SCFCdc4, containing CDC53,
SKP1 and the F-box protein CDC4 (Schwob et al., 1994;
Mathias et al., 1996; Feldman et al., 1997; Skowyra et al.,
1997). Conversely, CLN1-cyclin degradation is thought
to be mediated by UBC3 and a similar complex, SCFGrr1,
which, however, contains the alternative F-box protein
GRR1 (Barral et al., 1995; Willems et al., 1996; Li and
Johnston, 1997; Skowyra et al., 1997). We observed no
influence on the overall stability of CDC53 in rub1
mutants (not shown), suggesting that the modification of
CDC53 by RUB1 does not seem to target the protein
for proteasomal degradation. It is thus conceivable that,
analogously to SUMO-1, RUB1 may play a non-proteo-
lytic role. We speculate that the modification of CDC53
by RUB1 conjugation might, for example, influence SCF’s
subunit composition or activity, directing the complex
towards specific SCF substrates. In fact, we observed a
genetic interaction (synthetic lethality) of rub1, ula1, uba3
and ubc12 null mutants with ubc3/cdc34 temperature-
sensitive alleles (not shown), indicating that the RUB1
modification pathway is linked to the function of this cell-
division cycle protein. Fluorescence-activated cell sorter
(FACS) analysis of rub1 null mutants, however, has not
revealed any significant cell-cycle progression defect (not
shown). This suggests that RUB1-modified SCF, possibly
involving F-box proteins other than CDC4 or GRR1, may
act specifically on substrates whose stabilization in rub1
null mutants is not deleterious for cell growth or division.
Experiments designed to identify proteins which specific-
are currently underway and are expected to reveal insights
with respect to the cellular role of this novel protein
Materials and methods
Cloning and yeast techniques
The yeast techniques used here are described in Ausubel et al. (1994).
All strains are derivatives of DF5 (Finley et al., 1985; ura3-52 leu2-3,
-112 lys2-801 trp1-101 his3∆200). The RUB1 gene, including its intron
and 220 bp upstream and 360 bp downstream sequences, was cloned
into pUC19 (Ausubel et al., 1994) via PCR, creating plasmid pUCz48.
A rub1-1 null mutant was created by replacing the BglII fragment of
RUB1 by a double-stranded oligonucleotide, which introduces a
frameshift at codon 15. This manipulation results in the appearance of
a new stop at codon 16 and also in the introduction of an XbaI site. The
mutant allele was cloned into YIplac211 (Gietz and Sugino, 1988), and
the resulting plasmid YIrub1, which contains the URA3 marker, was
of RUB1 wild-type by the rub1-1 allele was performed by the two-step
gene replacement technique (Ausubel et al., 1994) as follows: the
plasmid YIrub1 was linearized at the unique BsaBI site within rub1-1
and transformed into DF5 diploids. These subsequently were sporulated
and, after tetrad dissection, ura?haploids were cultured and plated onto
5-fluoro-orotic acid (5-FOA) plates to select for eviction of the URA3
marker. 5-FOA-resistant haploids were then tested for retention of the
rub1-1 allele by PCR amplification of the RUB1 locus and digestion
of the product with XbaI. Positive clones were finally tested by
UBC12, including its intron, was cloned from a λEMBL3A library
using gene-specific probes and subcloned into pBluescript (Stratagene),
generating pKM050. A ubc12∆ deletion construct (pKM051) was made
by replacing the promoter region and 70% of the ORF with the TRP1
gene and was used for constructing a ubc12∆ strain (YDM3, MATa
ULA1 was cloned from genomic sequences by PCR and cloned into
pGEX2TK (pGEX-ULA); UBA3 was cloned by PCR into pBluescript
(pBS-UBA3) and pVL1392 (pVL-UBA3). A ula1∆ strain (YGD1, MATa
ula1::LEU2) was made by replacing the entire ORF by the LEU2 gene;
a uba3∆ strain (YGD4, MATa uba3::HIS3) was made by replacing 60%
of the coding region, including the sequence encoding the putative active
site, by the HIS3 gene. For thioester experiments, the RUB1-coding
sequence ending at codon 76 was cloned into pGEX2TK, generating
pGEXRUB. Genes encoding N-terminally triple HA-tagged versions of
RUB1 were placed under the control of the ADH1 promoter in YIplac204
(Gietz and Sugino, 1988). Plasmid KB622 was the source for epitope-
tagged CDC53 (gift of Daniel Kornitzer). The gene for CDC53, tagged
with both His6and an Xpress™ tag (Invitrogen) (HXCDC53), was cloned
into YIplac211 under the control of the GAL1-10 promoter. Genes for
HA-tagged versions of RUB1 were integrated into the TRP1 locus and
for tagged CDC53 into the URA3 locus.
For Western blots, yeast protein extracts were prepared by boiling cells
in Laemmli buffer. Standard techniques for SDS–PAGE and Western
blotting were used (Ausubel et al., 1994).
The RUB1 conjugate was isolated as follows: yeast spheroplasts from
a 9 l YPD culture of a haploid strain expressing HA-tagged RUB1 (see
above) were suspended in 18% Ficoll 400, 20 mM KPO4pH 6.45,
5 mM MgCl2and lysed in a Dounce homogenizer (Hurt et al., 1988).
The lysate was centrifuged at 13 000 g, and the pellet containing
organelles and intact nuclei was resuspended in 50 mM Tris–HCl pH 7.5,
1% Triton X-100, and applied to a DEAE column (Pharmacia). Bound
proteins were eluted with an NaCl gradient (0–300 mM). Fractions
containing RUB1 conjugates were then pooled, concentrated, incubated
with 1 ml of 12CA5 antibody-coupled protein A–Sepharose and eluted
by competition with 1 mg of HA peptide. The eluate was concentrated
and proteins were visualized by Coomassie Brilliant Blue staining after
SDS–PAGE (Figure 5A). A prominent band at ~100 kDa was excised
from the gel and subjected to MALDI mass spectrometry (Mortz
et al., 1994).
For thioester assays (Scheffner et al., 1993), RUB1 was obtained by
purifying a GST–RUB1 fusion [containing a thrombin cleavage and a
protein kinaseA (PKA) phosphorylationsite] from E.colicells expressing
pGEXRUB using a glutathione–Sepharose column. The fusion protein
was33P-labelled by PKA, cleaved with thrombin, thrombin was heat-
inactivated, and radiolabelled RUB1 was used for thioester assays. In
these assays, extracts of insect cells expressing UBA3, E.coli extracts
containing UBC12, and purified GST–ULA1 were used and incubated
in 25 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgCl2, 10 mM ATP and
0.1 mM dithiothreitol. The reaction products were analysed by non-
reducing SDS–PAGE followed by autoradiography.
We thank K.Ashman (EMBL, Heidelberg) for the identification of
CDC53 by mass spectrometry; D.Kornitzer, J.Dohmen and M.Scheffner
for providing strains and plasmids; H.Ulrich for designing Figure 2; and
M.Koegl and H.Ulrich for comments on the manuscript. This work was
supported by grants from the Deutsche Forschungsgemeinschaft and
Fonds der Chemischen Industrie to S.J.
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Received January 30, 1998; revised and accepted March 4, 1998