* Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, 72076 Tübingen, Germany; and ZMBH, Zentrum für
Molekulare Biologie, Universität Heidelberg, D-69120 Heidelberg, Germany
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Abstract |
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Ubiquitin-conjugating enzymes (UBC) catalyze the covalent attachment of ubiquitin to target proteins and are distinguished by the presence of a UBC domain required for catalysis. Previously identified members of this enzyme family are small proteins and function primarily in selective proteolysis pathways. Here we describe BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), a giant (528-kD) ubiquitin-conjugating enzyme from mice. BRUCE is membrane associated and localizes to the Golgi compartment and the vesicular system. Remarkably, in addition to being an active ubiquitin-conjugating enzyme, BRUCE bears a baculovirus inhibitor of apoptosis repeat (BIR) motif, which to this date has been exclusively found in apoptosis inhibitors of the IAP-related protein family. The BIR motifs of IAP proteins are indispensable for their anti-cell death activity and are thought to function through protein-protein interaction. This suggests that BRUCE may combine properties of IAP-like proteins and ubiquitin-conjugating enzymes and indicates that the family of IAP-like proteins is structurally and functionally more diverse than previously expected.
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Introduction |
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THE central importance of selective proteolytic systems in regulating cellular key events has been recognized recently. Progression through the eukaryotic cell cycle, for instance, is substantially regulated
through a timed and coordinated degradation of cyclins and inhibitors of cyclin-dependent protein kinases (for reviews see Hochstrasser, 1996; King et al., 1996
). Similarly,
the shift from one transcriptional or developmental program to another is often achieved through regulated destruction of regulatory proteins. Unlike several other posttranslational events (e.g., phosphorylation), proteolysis is
irreversible, and therefore proteolytic enzymes are usually
used for controlling unidirectional cellular pathways.
Selective degradation in eukaryotes primarily requires
the ubiquitin system that functions to mark proteins for
degradation by the multicatalytic protease, the proteasome (for reviews see Ciechanover, 1994; Jentsch and
Schlenker, 1995
; Hochstrasser, 1996
; Varshavsky, 1997
).
Proteins degraded by this pathway must first be recognized as substrates by components of the ubiquitin system.
A cascade of reactions catalyzed by several classes of enzymes is required to form an isopeptide bond between the
COOH terminus of ubiquitin and the
-amino group of a
lysine residue of an acceptor protein (for reviews see
Jentsch, 1992
; Ciechanover, 1994
; Hochstrasser, 1996
; Varshavsky, 1997
). Ubiquitin-activating (E1)1 enzyme hydrolyses ATP and forms a high-energy thioester between a
cysteine of its active site and the COOH terminus of ubiquitin. Activated ubiquitin is then passed on to ubiquitin-conjugating (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 ubiquitin-protein ligases (E3), which may possess substrate-binding properties (Scheffner et al., 1995
). Successive rounds of ubiquitination result in the formation of multiubiquitin chains
attached to proteolytic substrate proteins. Multiubiquitinated proteins are then recognized and degraded by the
proteasome.
E2 enzymes are thought to provide substrate specificity
to the proteolytic system and are encoded by large gene
families by apparently all eukaryotes. In the yeast Saccharomyces cerevisiae, this family comprises 11 members
(ubiquitin-conjugating enzymes [UBC]), which can be distinguished by their intracellular localization and cellular
functions (Jentsch, 1992; Varshavsky, 1997
). Known functions of the yeast E2s include DNA repair, cell cycle progression, sporulation, peroxisome biogenesis, and heat and
heavy metal tolerance (Jentsch, 1992
). Previously identified E2 enzymes are small proteins (16-35 kD) that bear a
conserved ~16-kD so-called UBC domain (Jentsch et al.,
1990
). The active-site cysteine residue required for the formation of a thioester-linked E2-ubiquitin complex is located within this domain. Some E2s consist solely of the
UBC domain, whereas others possess short COOH-terminal extensions. Among the known substrates are cyclins,
CDK inhibitors, transcription factors, subunits of trimeric
G proteins, and aberrant proteins (Hochstrasser, 1996
;
Varshavsky, 1997
). The majority of E2 enzymes are soluble proteins of the cytosol and the nucleus, but in yeast,
three E2s are known to localize to intracellular membranes. UBC10 is a peripheral membrane protein of peroxisomes and is required for the biogenesis of this organelle (Wiebel and Kunau, 1992
). UBC6 and UBC7
localize to the ER and are integral and peripheral membrane proteins, respectively. Both enzymes collaborate in
ER-associated degradation of short-lived and aberrant
proteins (Sommer and Jentsch, 1993
; Biederer et al., 1997
). From higher eukaryotes, however, no membrane-associated E2s have been reported previously.
Here we describe BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), a strikingly novel ubiquitin-conjugating enzyme from mouse. BRUCE is a giant 528-kD protein that, in contrast to previously identified ubiquitin-conjugating enzymes, is associated with the Golgi compartment and the vesicular system. We show that the UBC domain of BRUCE can be charged with ubiquitin in vitro, indicating that the protein possesses ubiquitin-conjugating activity. Remarkably, BRUCE also possesses a baculovirus inhibitor of apoptosis repeat (BIR) motif, a hallmark of apoptosis inhibitors of the IAP (inhibitor of apoptosis protein) class. This emphasizes the structural diversity of IAP-like proteins and suggests that its new member, BRUCE, may function through its ubiquitin-conjugating activity.
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Materials and Methods |
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DNA Techniques
Standard DNA techniques were used (Ausubel et al., 1994). Two degenerate primers (primers A and B from Matuschewski et al., 1996
) corresponding to conserved sequences of ubiquitin-conjugating enzymes were
used for amplification with genomic mouse DNA as a template. The obtained PCR fragment was used for screening a mouse brain cDNA library
(CLONTECH Laboratories, Palo Alto, CA). One positive clone was isolated and sequenced, and 5' sequences were used as a probe in a subsequent screen. After 11 similar rounds of screening, 15 overlapping cDNA
clones encompassing a sequence of 15,475 bp with a single open reading
frame (ORF) of 14,535 bp were each subcloned via the EcoRI site into
pUC19 (Ausubel et al., 1994
). All cDNA clones were sequenced at least
twice in both orientations using a sequenase kit (Amersham Corp., Arlington Heights, IL). Northern blots of mouse multiple tissue and mouse embryo (CLONTECH Laboratories) were probed with gene-specific 5'
(bp 195-828) or 3' sequences (bp 10865-15475), or with
-actin cDNA for
loading control. Hybridization was carried out according to the manufacturer's protocol.
Antibodies and Western Analysis
Two polypeptides corresponding to the NH2-terminal (amino acids 356- 493) and to the COOH-terminal (amino acids 4439-4845) part of BRUCE fused to the His6-tag sequence were expressed and purified by the QIAexpress System (Qiagen, Chatsworth, CA). After purification by SDS-PAGE, rabbits were immunized with the protein, and two polyclonal antibodies, "N" and "C" (corresponding to the NH2- and COOH-terminal fragments, respectively), were obtained. Antibodies against synaptophysin, MAP2 (both from Boehringer Mannheim Corp., Indianapolis, IN), TGN38, and TAU (kindly provided by G. Banting [University of Bristol, UK] and R. Brandt [University of Heidelberg], respectively) are mouse monoclonals, and anti-PDI antibodies are rabbit polyclonal (kindly provided by B. Dobberstein [University of Heidelberg]). Dichlorotriazinyl-fluoresceine (DTAF)- or lissamine rhodamine sulfonyl chloride (LRSC)-labeled and peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies were purchased from Dianova (Hamburg, Germany). For Western blots, the BRUCE protein of membrane fractions (see below) was separated by 4-14% gradient SDS-PAGE; all other proteins were separated by 12% SDS-PAGE and transferred to polyvinyl difluoride membranes.
Cell Fractionations
For the experiment shown in Fig. 3 B, rat PC12 cells were metabolically labeled with [35S]methionine and cracked in homogenization buffer (10 mM Hepes, pH 7.2, 0.25 M sucrose, 1 mM magnesium acetate, 1 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), and nuclei, organelles, and unbroken cells were pelleted by centrifugation at 1,000 g for 10 min. The postnuclear supernatant was centrifuged at 100,000 g for 60 min to obtain cytosolic and membrane fractions, and both fractions were adjusted to the same volume (yielding fractions S and P, Fig. 3 B, lanes 1 and 2). Two similarly prepared membrane fractions were further processed by extensive washes with either 0.5 M NaCl or PBS by passing through a 26-gauge needle, and the material was fractionated as above into soluble (S) and pellet (P) fractions (Fig. 3 B, lanes 3 and 4, and 5 and 6, respectively). BRUCE was detected in aliquots of these fractions by immunoprecipitation with antibodies C and N, and PDI and synaptophysin control proteins were detected by Western blotting. Immunoprecipitation of BRUCE with affinity-purified BRUCE-specific antibodies was carried out in a buffer containing 0.3% NP-40, 0.5 mM EDTA, 0.5 mM PMSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin for 1 h at 4°C. For the proteinase K sensitivity experiment shown in Fig. 3 C, postnuclear supernatant without protease inhibitors was incubated with or without 0.04 µg/ml proteinase K for 15 min on ice. After addition of protease inhibitors (2 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), membranes were pelleted by centrifugation at 100,000 g for 60 min, and BRUCE and PDI were detected by Western blot analysis.
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Confocal Microscopy
Rat neuroendocrine PC12 cells and primary rat hippocampal neurons
(kindly provided by A. Clement [University of Heidelberg]) were grown
on coverslips, washed once with PBS, fixed, and permeabilized with precooled methanol for 5 min at 20°C. After drying, cells were washed with
PBS and incubated with 10% goat serum for 20 min. Coverslips were incubated with the respective antibodies, washed, and stained with secondary
antibodies (LRSC-labeled goat anti-mouse or DTAF-labeled goat anti-
rabbit antibodies [1:200]) as described (Bos et al., 1993
). Confocal immunofluorescence microscopy was performed using a Leica TCS4D microscope
(Deerfield, IL) with a 63× 1.4 numerical aperture.
Thioester Assays
For thioester assays, ubiquitin was obtained by purifying a glutathione-
S-transferase-ubiquitin fusion (containing a thrombin-cleavage and a protein kinase A phosphorylation site; kindly provided by M. Scheffner [German Cancer Center, Heidelberg, Germany]) from Escherichia coli cells
expressing pGEXUBI using a glutathione-Sepharose column. The fusion
protein was radiolabeled using protein kinase A and [32P]ATP and
cleaved with thrombin, and thrombin was heat-inactivated. Radiolabeled
SUMO-1 and NEDD8 were generated by a similar procedure (Schwarz et
al., 1998
). E. coli BL21(DE3) cells transformed with plasmid pET3a
(Novagen, Madison, WI) expressing the COOH-terminal 406 amino acids
from BRUCE (FUBC) or cells transformed with plasmid pQE9 (Qiagen)
expressing an identical version NH2-terminally extended by additional six histidine residues (HFUBC) were generated. Extracts from these cells
were incubated for 10 min at 25°C in the presence of a reticulocyte lysate
(as a source for ubiquitin-activating activity; Stratagene, La Jolla, CA)
and radiolabeled ubiquitin (or ubiquitin-like proteins) in a buffer containing 25 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 10 mM ATP, and 0.1 mM DTT. Extracts from cells expressing UbcH7 ubiquitin-conjugating
enzyme were used as a positive control. The reaction products were incubated with sample buffer under nonreducing or reducing conditions (10 min boiling in the presence of 100 mM DTT) followed by SDS-PAGE and
autoradiography.
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Results |
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Identification of BRUCE and Expression Pattern
In the course of a homology-based screen for murine
genes encoding ubiquitin-conjugating enzymes (Matuschewski et al., 1996), we identified a DNA fragment that
hybridized to an exceptionally long mRNA. We obtained
15 overlapping complementary DNA (cDNA) clones after
12 rounds of screening, which identified a single large
ORF. The DNA sequence of this ORF (14,535 bp) predicts a giant protein with 4,845 amino acid residues (528 kD;
Fig. 1), which we termed BRUCE. Northern (RNA) analysis with probes corresponding to either NH2- or COOH-terminal sequences of BRUCE detected an identical ~15-kb
mRNA species, confirming that the isolated cDNAs corresponded to the same gene (Fig. 2). BRUCE is expressed in most tissues of adult mice, yet most prominently in the
brain and the kidney. However, total mRNA levels vary
strongly during mouse embryogenesis, possibly indicating
that controlled levels of BRUCE are important during development (Fig. 2). Similar mRNAs could be detected in
rat and man (not shown), indicating that BRUCE is ubiquitously expressed in mammals.
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Localization at Endomembranes
BRUCE is one of the largest proteins known to date, but
unlike most other giant proteins, it is nonrepetitive in
structure. The bulk of the protein exhibits no significant
sequence similarity to other known proteins and displays a
high percentage (~35%) of hydrophobic amino acid residues. We therefore speculated that the protein might be
membrane bound. Antibodies raised against NH2-terminal
and COOH-terminal domains of BRUCE, respectively, identified an ~500-kD protein in cell extracts, and fractionation studies indeed established a localization of the
protein chiefly in membrane fractions (Fig. 3, A and B).
The protein was extractable by salt, indicating that
BRUCE is membrane associated but not embedded into
the lipid bilayer (Fig. 3 B). As indicated by proteinase K
sensitivity assays, the protein appears to localize to the cytosolic face of the membrane (Fig. 3 C). Interestingly, confocal microscopy identified BRUCE in a punctated cytosolic distribution and in a Golgi-related compartment,
specifically the TGN, where the protein colocalized with
TGN38 (Bos et al., 1993; Humphrey et al., 1993
), a TGN-specific marker protein (Fig. 3 D). To refine the characterization of the intracellular distribution of BRUCE, we took
advantage of the highly polarized organization of neuronal
cells (Parton and Dotti, 1993
). To this end, primary neurons were prepared and stained for BRUCE with affinity-purified BRUCE-specific antibodies. In these cells, besides its localization in the TGN of the cell body, BRUCE
was also detectable in a dotted distribution in axons and
dendrites, indicating that the protein is additionally associated with more distal vesicular structures (Fig. 3 D).
BRUCE Is Related to Ubiquitin-conjugating Enzymes
The predicted primary sequence indicates that BRUCE
harbors a typical UBC domain close to the protein's
COOH terminus (Fig. 1). UBC domains are hallmarks of
E2 enzymes of the ubiquitin-dependent proteolytic system
and related pathways (Jentsch et al., 1990). UBC domains
are specific for E2 enzymes and carry the active-site cysteine residue required for thioester formation (Jentsch et al.,
1990
; and see introduction). It has been shown recently, however, that UBC domains are also found in E2 enzymes, which mediate the conjugation of ubiquitin-like
proteins (Johnson and Blobel, 1997
; Liakopoulos et al.,
1998
; Schwarz et al., 1998
). The UBC domain of BRUCE
is ~37% identical to those of other ubiquitin-conjugating enzymes and harbors a putative active-site cysteine residue at a conserved position (Fig. 4 A).
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Ubiquitin-conjugating Activity of BRUCE
Because of the presence of a UBC domain, we speculated
that BRUCE possesses ubiquitin-conjugating activity. To
test this hypothesis, we asked whether the protein can be
charged in vitro with ubiquitin in a reaction depending on
ubiquitin-activating enzyme, E1. Because of the size of the
protein and the absence of a cDNA clone encompassing the complete 14,535 bp of the BRUCE open reading
frame, we expressed COOH-terminal fragments of
BRUCE in E. coli for enzyme assays. Two variants were
constructed, one corresponding to the COOH-terminal 406 amino acid residues of BRUCE containing its complete UBC domain, and a similar version, extended by an
additional poly-histidine tag. Bacterial protein extracts expressing either of the two fragments were assayed in vitro
for thioester formation of the recombinant polypeptides
with radiolabeled ubiquitin in the presence of E1 activity
and ATP. Only in the presence of these bacterial extracts (Fig. 4 B) were additional radiolabeled protein complexes
with sizes of ~55 and ~60 kD formed, consistent with the
sizes of adducts between ubiquitin and the shorter or
longer fragments of BRUCE's UBC domain, respectively
(Fig. 4 B, DTT, lanes 4 and 5). Complex formation depended on ATP and the presence of E1 activity. The complexes were sensitive to boiling under reducing conditions, indicating that the protein fragments are indeed linked via
a thioester bond (Fig. 4 B, +DTT, lanes 4 and 5). This reaction was specific for ubiquitin, as neither SUMO-1 nor
NEDD8, two mammalian ubiquitin-like proteins (Kumar
et al., 1992
; Johnson and Hochstrasser, 1997
), were able to
form adducts with the UBC domain of BRUCE under
similar conditions (data not shown).
BRUCE Is Related to IAP Apoptosis Inhibitors
Intriguingly, database searches identified in addition to
the UBC domain a single BIR repeat close to the NH2 terminus of the BRUCE protein. BIR repeats are cysteine
rich, putative zinc-binding domains of about 70 amino acid
residues in length, which to this date have exclusively been
found in IAP-related apoptosis inhibitors. Members of the
family of IAP-related cell death inhibitors are characterized by the presence of up to three NH2-terminally positioned BIR repeats, followed by distinct COOH-terminal
sequences (Birnbaum et al., 1994; Hay et al., 1995
; Rothe
et al., 1995
; Roy et al., 1995
; Ambrosini et al., 1997
; Clem
and Duckett, 1997
). Previously known members fall into
three classes, distinguished by their protein domain structure. The first class is represented by the anti-apoptotic
IAP proteins of baculovirus, insects, and mammals, which
are small proteins with up to three NH2-terminal BIR repeats, linked to a COOH-terminal RING finger (Birnbaum et al., 1994
; Hay et al., 1995
; Rothe et al., 1995
; Clem
and Duckett, 1997
). The second class is defined by neuronal apoptosis inhibitor protein (NAIP), a human 140-kD
protein with three NH2-terminal BIR repeats and a long
COOH-terminal tail that lacks a RING finger motif (Roy et al., 1995
; Liston et al., 1996
). Survivin, a small human
cell death inhibitor, which essentially consists of a single
BIR motif, defines the third class (Ambrosini et al., 1997
).
The BIR motif of BRUCE is ~40% identical in sequence to those of other IAP relatives and bears all known consensus elements of this domain, including a characteristic cysteine/histidine motif, which is thought to function in zinc binding (Fig. 5). The structure of BRUCE is unprecedented, however, as the protein has a single BIR repeat (similar to survivin) plus an exceptionally long tail, which exhibits no homology to the extensions of previously known IAP relatives, i.e., the tail of NAIP and the RING fingers of the IAP proteins. BRUCE is the first example of an IAP-like protein with a known enzymatic activity. Interestingly, the BIR motifs of BRUCE and survivin are unique since they contain an insertion of three amino acid residues (one of which is a proline residue) in between the two conserved halves of the BIR motif, which we term the FY (rich in phenylalanine and tyrosine residues) and the CH (rich in cysteine and histidine residues) regions, respectively. In the mouse genome, the regions encoding the FY and CH domains of BRUCE are separated by an intron (Pyrowolakis, G., and S. Jentsch, unpublished results). This suggests that the complete BIR motif has probably evolved by exon shuffling and that the FY and CH may represent functionally distinct folding domains separated by a linker sequence.
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Discussion |
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Apoptotic cell death is a highly regulated process of multicellular organisms through which superfluous or harmful
cells are eliminated (for review see White, 1996). During
this process, apoptotic cells go through sequential steps of
disintegration that include chromatin cleavage, organelle
breakdown, and the fragmentation of cellular material
into membrane-surrounded vesicles that are rapidly ingested by neighboring cells. Cell death can be triggered by
a variety of stimuli, but once initiated, apoptosis appears
to proceed through a common pathway. The basic cell
death machine comprises a family of related cysteine proteases, termed caspases, which cleave proteins COOH-terminal of aspartate residues (for review see Salvesen and
Dixit, 1997
). Caspases are synthesized as inactive proenzymes that are activated by proteolytic cleavage. The processing sites themselves match the consensus cleavage
specificity of caspases, and it is thus assumed that the apoptotic suicide program is orchestrated through a cascade of
auto- and trans-cleavages of different caspases. Activated
caspases can cleave further targets, including components
of the cytoskeleton, nuclear lamins, poly(ADP-ribose) polymerase, DNA-dependent protein kinase, and inhibitor
of caspase-activated DNase (Salvesen and Dixit, 1997
;
Enari et al., 1998
; Sakahira et al., 1998
). The cleavage of
these downstream targets is thought to execute the final cell
death program with the characteristic morphological changes.
The cellular suicide program can be blocked by a variety
of proteins, and many of these factors are known to interfere with caspase function. Bcl-2, a membrane protein of
primarily the outer membrane of mitochondria, appears to
inhibit apoptosis by blocking the efflux of cytochrome c
from mitochondria (White, 1996; Reed, 1997
). This in turn
is thought to impede caspase-9 activation because maturation from its inactive proenzyme form is stimulated by cytochrome c in conjunction with the Apaf-1 protein (Li et al.,
1997
). In contrast, direct inhibition of caspase function is
achieved by the cell death inhibitor p35 from baculovirus, which seems to act through caspase binding (Clem et al.,
1991
; Bump et al., 1995
; Xue and Horvitz, 1995
).
Another group of anti-apoptotic proteins is the IAP
family of apoptosis inhibitors (for review see Clem and
Duckett, 1997). Members of this protein family are distinguished by the presence of up to three BIR motifs. BIR
motifs are required, and often sufficient, for the anti-apoptotic function of IAP-related proteins and are known to
act through protein-protein interactions (Clem and Duckett, 1997
). Several binding proteins have been found previously, which include TRAF signaling molecules (Rothe et al.,
1995
) and the proteins Doom and Reaper from flies (Harvey et al., 1997
; Vucic et al., 1997
). Recent data indicated
that a principal anti-apoptotic function of BIR-containing
proteins may be their ability to bind and inhibit specific
caspases (Devereaux et al., 1997
; Roy et al., 1997
; Seshagiri and Miller, 1997
).
In this paper we identify BRUCE, an unusual membrane-associated protein from mouse that combines properties of IAP-like proteins with ubiquitin-conjugating enzymes. Although the cellular function of BRUCE is not known at present, the intriguing modular design of the protein with an NH2-terminal BIR repeat and an active ubiquitin-conjugating enzyme domain at the protein's COOH terminus suggests a role in coupling anti-apoptosis pathways to the ubiquitin/proteasome proteolytic machinery. It seems attractive to speculate that BRUCE may be regulated by specific BIR motif-binding proteins or, alternatively, may target these proteins (e.g., caspases) for degradation. In contrast to a possibly reversible inhibition of caspases by other IAP relatives, proteolytic inactivation of apoptosis regulators would allow an irreversible shut-down of specific cell death pathways.
BRUCE is structurally strikingly different from previously known E2 enzymes, which are small proteins (14-34
kD) that either consist of the UBC domain alone (class I)
or possess additional short COOH- (class II) or NH2-terminal (class III) extensions (Jentsch et al., 1990; Matuschewski et al., 1996
). BRUCE, which bears a long NH2-terminal and a short COOH-terminal extension, defines a
new class of E2 enzymes, termed class IV. Interestingly, the overall domain structure of BRUCE resembles more a
class of E3 enzymes (ubiquitin ligases) of the ubiquitin system that possess COOH-terminal, so-called HECT domains involved in thioester formation with ubiquitin
(Huibregtse et al., 1995
; Scheffner et al., 1995
, Rosa et al.,
1996
). These E3 enzymes typically bear within their NH2-terminal domains binding sites for proteolytic substrates and for cofactors that alter the enzyme's substrate specificity (Huibregtse et al., 1995
; Scheffner et al., 1995
). This
modular structure enables the enzyme to bind substrates
directly and to catalyze their ubiquitination via its COOH-terminal catalytic domain. It is conceivable that BRUCE
may function analogously and that the BIR motif of
BRUCE constitutes either a binding site for a substrate or
a regulator.
BRUCE also differs from previously known E2 enzymes with respect to its intracellular localization.
BRUCE is the first example of a membrane-associated E2
enzyme from mammalian cells, and its localization at a
Golgi-related compartment and the vesicular system is unprecedented for this enzyme class. Remarkably, only one
other enzyme of the ubiquitin system has been detected at a Golgi-related compartment previously. This is p532, a
very large putative E3 ubiquitin ligase with a COOH-terminal HECT domain and an expression pattern similar to
BRUCE (Rosa et al., 1996). This enzyme bears several
WD and RCC1 repeats, binds clathrin, and stimulates guanine nucleotide exchange on ARF1 and Rab proteins, which are important for membrane fusion and trafficking
(Rosa et al., 1996
; Rosa and Barbacid, 1997
). How this
function is mechanistically tied to the ubiquitin system is
not known at present, but it will be important to test
whether BRUCE as an E2 enzyme can charge this large
putative E3 enzyme with ubiquitin. If so, it seems attractive to speculate that their cooperative function is linked to the membrane fusion events that occur during apoptosis. Further studies require the assembly of the complete
~15-kb cDNA of BRUCE into a single clone for gene
transfer studies, which are expected to answer if and how
BRUCE is involved in preventing apoptosis.
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Footnotes |
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Received for publication 6 April 1998 and in revised form 27 April 1998.
The work by H.-P. Hauser was done in the former laboratory of S. Jentsch in Tübingen. We thank T. Hoppe for important contributions to this study; M. Scheffner, G. Banting, R. Brandt, B. Dobberstein, and A. Clement for providing materials; M. Scheffner for help and advice for thioester experiments; F. Weinreich for help with DNA sequencing; E. Löser and P. Hubbe for technical assistance; and H. Ulrich for comments on the manuscript.This work was supported by grants of the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie to S. Jentsch.
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Abbreviations used in this paper |
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BIR, baculovirus inhibitor of apoptosis repeat; BRUCE, BIR repeat containing ubiquitin-conjugating enzyme; E1, ubiquitin-activating enzyme; IAP, inhibitor of apoptosis protein; NAIP, neuronal apoptosis inhibitor protein; ORF, open reading frame; UBC, ubiquitin-conjugating enzyme.
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