BRUCE interacts non-covalently with UBA6 and FAT10

We decided, that the huge membrane- associated and evolutionary highly conserved protein BRUCE (528 kDa) is a very interesting protein to investigate, as it contains a catalytic ubiquitin conjugating enzyme (UBC) domain at its C-terminus, and a BIR (baculovirus inhibitor of apoptosis repeat) motif. BRUCE has been previously described to act as a chimeric ubiquitin E2/E3 ligase with Smac being a substrate (Bartke et al., 2004).

Furthermore, during final stages of cytokinesis BRUCE moves from the vesicular system to the midbody ring and serves as a platform for the membrane delivery machinery and mitotic regulators, thereby ensuring controlled abscission (Pohl and Jentsch, 2008).

88 Moreover, due to its N-terminal BIR domain, which are found within inhibitor of apoptosis proteins (IAP), it can further act as an antiapoptic IAP (Bartke et al., 2004).

Therefore, BRUCE may function to mediate both, ubiquitin-dependent proteolysis and contribute to anti-apoptotic cellular pathways.

To verify the yeast-two-hybrid findings and confirm the interaction with UBA6, we examined the ability of human UBA6 to interact with the human native full-length BRUCE (kindly provided from S. Jentsch) in human cells. The putative association of UBA6 with BRUCE might argue for an UBE1- independent loading of BRUCE with either ubiquitin of FAT10 by this newly identified ortholog. Hence, we tested the possibility of interaction between BRUCE and either ubiquitin or FAT10.

FLAG-tagged BRUCE together with HA-tagged FAT10, -Ubiquitin and -UBA6 were co-expressed in HEK293T cells. After 24 h ectopic protein expression, cells were washed twice in cold PBS and scraped on ice from cell culture dishes. Cells were instantly lysed in ice-cold BRUCE-IP buffer (Table 14) containing complete protease inhibitor mix and lysed on ice for 30 min. Whole cell lysate were boiled without ß-mercaptoethanol (non-reducing conditions) to preserve thioester linkages or in Laemmli buffer containing 10% ß-mercaptoethanol (reducing conditions), to cleave thioester bonds. Samples were separated on 4-12 % Bis-TRIS SDS-gels and directly immunoblotted with a anti-HA-antibody to analyze expression of HA-tagged ubiquitin, FAT10 or UBA6. Following immunoprecipitation with anti-FLAG-M2-conjugated agarose against the FLAG-tag of BRUCE, samples were boiled in Laemmli buffer without or with 10 % ß-mercaptoethanol and subjected to SDS-PAGE and Western blot analysis using a anti-HA antibody.

Results

89 (a)

(b)

90

Figure 16: UBA6, ubiquitin and FAT10 co-immunoprecipitate with full length BRUCE in HEK293 cells.

(a) 293T cells were transiently transfected with FLAG-BRUCE, HA-UBA6, HA-FAT10 and HA-Ubiquitin as indicated. (a) After 24 h ectopic protein expression whole cell lysates were boiled in Laemmli buffer without (non-reducing conditions) or with 10 % ß-mercaptoethanol ((non-reducing conditions) and separated on 4-12 % Bis-Tris gels and analyzed by Western blotting with a HA-reactive monoclonal antibody. (b) After immunoprecipitation against the FLAG-tag of BRUCE with anti-FLAG-M2-conjugated agarose, samples were boiled in Laemmli buffer without (non-reducing conditions) or with 10 % ß-mercaptoethanol (reducing conditions) and separated on 4-12% Bis-Tris SDS gels and subjected to Western blot analysis with a HA-reactive monoclonal antibody.

Expression of HA-tagged UBA6 (~110 kDa), FAT10 (18 kDa), ubiquitin (8 kDa) and ubiquitin conjugated proteins could be detected under non-reducing as well as under reducing conditions (Figure 16 (a)). After immunoprecipitation against the FLAG-tag of FLAG-BRUCE, and Western blot analysis with a HA-reactive antibody an upcoming ubiquitin ladder was visible under non-reducing and reducing conditions, suggesting that BRUCE interacts with ubiquitin or ubiquitin conjugates (Figure 16 (b), lane 3+4). In contrast, a non-covalent interaction of FLAG-BRUCE and HA-UBA6 as well as with HA-FAT10 could be detected by Western blot analysis under reducing conditions (Figure 16 (b) lane 6, 7 and 9), whereas under non-reducing conditions only a slight band was apparent at the height of UBA6 and almost no FAT10 was detectable, suggesting that BRUCE interacts with UBA6 and FAT10 via a thioesterbond linkage.

To detect conjugation bands at the height of 500-600 kDa 3-8 % Tris-Acetate gels for large protein separation were used.

(a)

Results

91

Figure 17: FAT10 becomes thioester-linked to full length BRUCE in HEK293 cells.

(a) 293T cells were transiently transfected with FLAG-BRUCE, HA-UBA6, HA-FAT10 and HA-Ubiquitin as indicated. (a) After 24 h ectopic protein expression immunoprecipitation was performed with anti-FLAG-M2 affinity gel. Samples were boiled in Laemmli buffer without (non-reducing) or with 10 % ß-mercaptoethanol (reducing) and were separated on 3-8 % Tris-Acetate gels followed by Western blot analysis using a FLAG-reactive antibody to reveal FLAG-BRUCE expression. (b) After immunoprecipitation against the FLAG-tag of BRUCE with anti-FLAG-M2-conjugated agarose, samples were boiled in Laemmli buffer without (non-reducing conditions) or with 10 % ß-mercaptoethanol (reducing conditions) and separated on 4-12% Bis-Tris SDS gels and subjected to Western blot analysis with a HA-reactive monoclonal antibody. Arrow head indicates thioester-linked BRUCE conjugates.

FLAG-BRUCE expression could be detected at a molecular weight of about 530 kDa (Figure 17 (a)). Interestingly, after immunoprecipitation against the FLAG-tag of BRUCE and immunoblotting with a FLAG-reactive antibody, double bands at the height of BRUCE appear under non-reducing (Figure 17 (a), arrow head), but not under reducing conditions (Figure 17 (a), reducing) which indicates that BRUCE becomes modified via a thioester-linkage to FAT10. Moreover, at the size of BRUCE (~530 kDA) very weak conjugate bands were observable after immunoprecipitation against the FLAG-tag of BRUCE and Western blot analysis with a HA-reactive monoclonal antibody, when BRUCE and HA-UBA6, FLAG-BRUCE and HA-FAT10 or even when all three proteins were overexpressed under non-reducing conditions (Figure 17 (b), lane 6,7 and 9). Due to the very faint band appearing, further experiments are required to ensure that BRUCE gets modified with FAT10 via a thioester-linkage.

(b)

92 The conjugate bands almost completely disappeared under reducing (10% ß-mercaptoethanol) conditions, which additionally argue for a thioester-bond between the respective proteins. Besides, an increased amount of co-immunoprecipitated HA-UBA6 is detectable under reducing condition, which strengthens the hypothesis that BRUCE might become thioester-linked to UBA6 (Figure 17 (b), lane 6+7).