A giant ubiquitin-conjugating enzyme related to IAP apoptosis inhibitors

Abstract

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.

Figure 3

The BRUCE protein is associated with endomembranes. (A) Western blot of membrane fractions from mouse brain, mouse AtT20 (neuroendocrine), mouse N2a (neuroblastoma), and rat PC12 (neuroendocrine) cell lines probed with BRUCE-specific antibodies (antibody C). (B) Immunoprecipitation of in vivo–radiolabeled proteins with BRUCE-specific antibodies (antibodies C and N) from cytosolic (S, supernatant) and membrane (P, pellet) fractions (see Materials and Methods). BRUCE is enriched in membrane fractions (see below) and can be extracted by 0.5 M NaCl (compare lanes S and P) or buffer (PBS; compare lanes S and P). Antibodies specific for the ER-lumenal protein disulfide isomerase (PDI) and synaptophysin, an integral membrane protein of synaptic vesicles, were used for detection by Western blotting from the identical protein fractions. These proteins used as controls were found in the pellet fraction. A portion of lumenal PDI was also detectable in the cytosolic fraction (S, first lane), presumably because some microsomal vesicles were “inside-out” or disintegrated. (C) In contrast to lumenal PDI, BRUCE is sensitive to proteinase K treatment at membranes in vitro, indicating that BRUCE faces to the cytosol. (D) Confocal micrographs of rat PC12 cells and primary rat neurons stained with antibodies (indirect immunofluorescence) specific for BRUCE (green, antibodies C and N), and the marker proteins (red) TGN38 (marker for TGN; Humphrey et al., 1993), MAP2 (marker for dendrites), and TAU (marker for axons). Note that BRUCE appears in a punctated cytosolic distribution and is present in the TGN, axons, and dendrites. Colocalization of BRUCE with marker proteins is shown by superimposition (yellow). The BRUCE-specific antibodies used in these studies (A–D) are affinity-purified, and the signals are specific since they could be completely eliminated by competition with epitope-containing protein fragments (not shown).

Figure 3

The BRUCE protein is associated with endomembranes. (A) Western blot of membrane fractions from mouse brain, mouse AtT20 (neuroendocrine), mouse N2a (neuroblastoma), and rat PC12 (neuroendocrine) cell lines probed with BRUCE-specific antibodies (antibody C). (B) Immunoprecipitation of in vivo–radiolabeled proteins with BRUCE-specific antibodies (antibodies C and N) from cytosolic (S, supernatant) and membrane (P, pellet) fractions (see Materials and Methods). BRUCE is enriched in membrane fractions (see below) and can be extracted by 0.5 M NaCl (compare lanes S and P) or buffer (PBS; compare lanes S and P). Antibodies specific for the ER-lumenal protein disulfide isomerase (PDI) and synaptophysin, an integral membrane protein of synaptic vesicles, were used for detection by Western blotting from the identical protein fractions. These proteins used as controls were found in the pellet fraction. A portion of lumenal PDI was also detectable in the cytosolic fraction (S, first lane), presumably because some microsomal vesicles were “inside-out” or disintegrated. (C) In contrast to lumenal PDI, BRUCE is sensitive to proteinase K treatment at membranes in vitro, indicating that BRUCE faces to the cytosol. (D) Confocal micrographs of rat PC12 cells and primary rat neurons stained with antibodies (indirect immunofluorescence) specific for BRUCE (green, antibodies C and N), and the marker proteins (red) TGN38 (marker for TGN; Humphrey et al., 1993), MAP2 (marker for dendrites), and TAU (marker for axons). Note that BRUCE appears in a punctated cytosolic distribution and is present in the TGN, axons, and dendrites. Colocalization of BRUCE with marker proteins is shown by superimposition (yellow). The BRUCE-specific antibodies used in these studies (A–D) are affinity-purified, and the signals are specific since they could be completely eliminated by competition with epitope-containing protein fragments (not shown).

Figure 1

Primary structure of BRUCE predicted from the cDNA sequence. (Sequence data are available from GenBank/EMBL/ DDBJ under accession number Y17267.) The NH₂-terminally positioned single BIR repeat and the UBC domain close to the COOH terminus are highlighted. The position of the presumed active-site cysteine residue required for thioester formation with ubiquitin is marked within the UBC domain. The presumed initiator methionine residue was deducted from the position of the first ATG codon found in the cDNA sequence. A schematic representation of the domain structure of BRUCE is shown at the bottom.

Figure 2

Levels of BRUCE transcripts. Northern blots of oligo(dT)-purified mRNA from multiple tissues (left) and from total mouse embryos (right; days 7–17 postconception; both blots from CLONTECH Laboratories). Note that 3′- (bp 10865– 15475), as well as 5′-probes (bp 195–828) recognize the ∼15-kb transcript of BRUCE. The transcript is particularly abundant in brain and kidney, and levels vary during development. Actin mRNA was used for loading control. (The lower bands represent muscle-specific isoforms.)

Figure 4

BRUCE is a ubiquitin-conjugating enzyme. (A) Sequence comparison of the UBC domain of BRUCE (amino acids 4601–4671) with those of other ubiquitin-conjugating enzymes (S. cerevisiae UBC1, -2, -3, -5, -6, and -7; mouse UbcM2; human UbcH7 and -8; and Drosophila UbcD1). Residues identical in at least four domains (majority) are shaded. The position of the presumed active-site cysteine residue required for thioester formation with ubiquitin is marked by an asterisk. (B) Thioester complex formation between ubiquitin and fragments of BRUCE (^FUBC, ^HFUBC) consisting of the COOH-terminal 406 amino acid residues of BRUCE, which include the complete UBC domain. Assay reactions contained ATP, ³²P-labeled ubiquitin, reticulocyte lysate as a source for E1 activity, and crude extracts from bacteria expressing ^FUBC, ^HFUBC, or UbcH7 (Nuber et al., 1996; human ubiquitin-conjugating enzyme as positive control). Reactions were analyzed by SDS-PAGE under nonreducing (−DTT) or reducing (+DTT) conditions (Nuber et al., 1996). Note that only in the presence of ^FUBC or ^HFUBC were additional radiolabeled complexes with sizes of ∼55 and ∼60 kD formed (−DTT, lanes 4 and 5), consistent with the sizes of adducts between ubiquitin and ^FUBC or ^HFUBC, respectively. These complexes are thioester-linked since they disappear after boiling under reducing conditions (+DTT, lanes 4 and 5). Control reactions containing labeled ubiquitin incubated with either bacterial extract (lane 1), reticulocyte lysate (lane 2), or reticulocyte lysate plus extracts of bacteria harboring the vector plasmid (lane 3) are shown. Complex formation of ubiquitin with added UbcH7 (lane 6) or UbcH7 present in the reticulocyte lysate (lanes 2–5) is used as positive control. Bands corresponding to free ³²P-labeled ubiquitin and the respective thioester adducts are indicated.

Figure 4

BRUCE is a ubiquitin-conjugating enzyme. (A) Sequence comparison of the UBC domain of BRUCE (amino acids 4601–4671) with those of other ubiquitin-conjugating enzymes (S. cerevisiae UBC1, -2, -3, -5, -6, and -7; mouse UbcM2; human UbcH7 and -8; and Drosophila UbcD1). Residues identical in at least four domains (majority) are shaded. The position of the presumed active-site cysteine residue required for thioester formation with ubiquitin is marked by an asterisk. (B) Thioester complex formation between ubiquitin and fragments of BRUCE (^FUBC, ^HFUBC) consisting of the COOH-terminal 406 amino acid residues of BRUCE, which include the complete UBC domain. Assay reactions contained ATP, ³²P-labeled ubiquitin, reticulocyte lysate as a source for E1 activity, and crude extracts from bacteria expressing ^FUBC, ^HFUBC, or UbcH7 (Nuber et al., 1996; human ubiquitin-conjugating enzyme as positive control). Reactions were analyzed by SDS-PAGE under nonreducing (−DTT) or reducing (+DTT) conditions (Nuber et al., 1996). Note that only in the presence of ^FUBC or ^HFUBC were additional radiolabeled complexes with sizes of ∼55 and ∼60 kD formed (−DTT, lanes 4 and 5), consistent with the sizes of adducts between ubiquitin and ^FUBC or ^HFUBC, respectively. These complexes are thioester-linked since they disappear after boiling under reducing conditions (+DTT, lanes 4 and 5). Control reactions containing labeled ubiquitin incubated with either bacterial extract (lane 1), reticulocyte lysate (lane 2), or reticulocyte lysate plus extracts of bacteria harboring the vector plasmid (lane 3) are shown. Complex formation of ubiquitin with added UbcH7 (lane 6) or UbcH7 present in the reticulocyte lysate (lanes 2–5) is used as positive control. Bands corresponding to free ³²P-labeled ubiquitin and the respective thioester adducts are indicated.

Figure 5

Sequence comparison of the BIR motif of BRUCE (amino acids 261–333) with those from other IAP relatives. Residues identical in at least four motifs (majority) are shaded. Individual BIR repeats found in a single protein are numbered (I–III). The BIR motif of BRUCE is ∼40% identical in sequence to those of other IAP relatives, with a conserved FY (left part) and CH region (right part; see text).