5.1 The characterisation of the FAT10 p62 interaction
5.1.6 The isolated PB1 domain of HA-p62 doesn’t suffice to bind to
5.1.6.1 Transient transfection and co-immunoprecipitation experiments
Since those HA-p62 deletion constructs in which the deletion encompass the PB1 domain, the TRAF domain, or the N-terminal PEST domain were not FAT10ylated anymore (fig.20-21), it was tested whether these isolated p62 domains would suffice to become FAT10ylated. Therefore, HEK293T cells were transiently co-transfected with the HA-tagged constructs encoding for the respective isolated p62 domains and Flag-FAT10 (fig.30, lanes 9, 10, 11). As positive control, a sample of cells which co-expressed both Flag-FAT10 and HA-p62 was taken along (fig.30, lanes 8). Since the depletion of the UBA domain did not influence the FAT10ylation of HA-p62 (Aichem, Kalveram et al. 2012), the isolated UBA domain of p62 was taken along as a negative control (fig.30, lanes 7, 12). Further, Flag-FAT10 (fig.30, lanes 2, 14), HA-p62 (fig.30, lanes 3, 15), HA-HA-p62(PB1 only) (fig.30, lanes 4, 16), HA-HA-p62(TRAF only) (fig.30, lanes 5, 17), HA-p62(PEST only) (fig.30, lanes 6, 18) and HA-p62(UBA only) (fig.30, lanes 7, 19) were transfected alone as negative controls. As a mock control, cells were treated with the transfection reagent only (fig.30, lane 1, 13). An anti-Flag immunoprecipitation was performed in order to investigate whether the isolated p62 domains would be co-immunoprecipitated with FAT10 (fig.30, lanes 13-24). The samples were analysed by western blot experiments. In order to detect the monomeric HA-p62 and the isolated HA-p62 domains or the monomeric Flag-FAT10 as well as the Flag-FAT10 conjugates which were utilised for the anti-Flag immunoprecipitation experiments, the western blots with the cleared cell lysates (load) were stained with either anti-HA or anti-Flag antibodies respectively. The western blots of the anti-Flag-immunoprecipitation experiment were stained with either anti-Flag or anti-HA antibodies in order to detect either the immunoprecipitated monomeric Flag-FAT10 and the Flag-FAT10-conjugates, or the co-immunoprecipitated HA-p62 as well as the isolated HA-p62 domains. In the anti-HA western blot of the immunoprecipitation experiments only those anti-HA-p62 proteins should be detectable which are covalently attached to Flag-FAT10 or which formerly did interact non-covalently with Flag-FAT10.
Results
Figure 30: The isolated PB1 domain of p62 doesn’t seem to interact with Flag-FAT10, neither covalently nor non-covalently. HEK293T cells were transiently transfected with Flag-FAT10 and HA-p62 or the isolated HA-tagged p62 domains (PB1, TRAF, PEST, UBA) as indicated. After harvesting, the cleared supernatants were used for the co-immunoprecipitation experiments with anti-FLAG-M2-conjugated agarose. The samples were boiled with a 10 % β-mercaptoethanol containing SDS-sample buffer and separated by TRIS-Tricin gel electrophoresis (Schagger and von Jagow 1987). After the wet-blotting on PVDF membranes, the blots were probed with either anti-Flag, or anti-HA reactive antibodies. The monomeric wild-type HA-p62 has a molecular weight of 70 kDa, the HA-PB1 domain of 12,5 kDa, the TRAF domain of 4,5 kDa, the N-terminal PEST domain as well as the HA-UBA domain of 6 kDa and the monomeric FAT10 of 25 kDa. One experiment out of two independent experiments is shown.
In the anti-Flag and anti-HA stained western blots of the cleared cell lysates, Flag-FAT10 (fig.30, lanes 2, 8-12) and HA-p62 (fig.30, lanes 3, 8) were detectable in the samples which were transfected with the respective DNA constructs. Although being cloned into the same backbone plasmid and besides the verification of the cloning success by sequencing, out of the four isolated p62 domains only the HA-PB1 domain of p62 was detectable in the anti-HA western blot (fig.30, lanes 4, 9). In the anti-Flag western blots, of the corresponding anti-Flag immunoprecipitation experiment, the immunoprecipitated Flag-FAT10 was detectable for all Flag-FAT10 containing samples (fig.30, lanes 14, 20-24). In the anti-HA western blot of the
Results
immunoprecipitation experiments of the Flag-FAT10 and HA-p62 co-expressing sample (fig.30, lanes 21) as well as in the HA-p62 only containing sample (fig.30, lanes 15), indicating that it stacked to the anti-Flag coupled agarose unspecifically.
5.1.6.2 In vitro transcription/translation and GST pulldown experiments
Since, besides the HA tagged PB1 domain of p62 (fig.30, lane 4, 9), the isolated HA-p62 domains were not detectable in the total cell lysates of the transfected HEK293T cells (fig.30), the in vitro transcription and translation system was used in order to express HA-p62 (fig.31, lane 1) and the isolated HA-p62 domains (fig.31, lanes 4, 7, 10, 13) in the next experiment. In the subsequent GST pulldown with GST-FAT10, their non-covalent interaction capabilities with FAT10 were tested.
As negative controls, the GST pulldowns for HA-p62 and all isolated HA-p62 domains were additionally performed with recombinant GST protein instead of GST-FAT10.
The samples were separated in western blot experiments and the [35S] methionine labelled HA-p62 domains were detected by autoradiography and the GST-fusion proteins were visualised by Coomassie Brilliant Blue-staining.
Results
Figure 31: The isolated PB1 domain of p62 doesn’t seem to interact with Flag-FAT10 non-covalently. The wild-type HA-p62 and the isolated HA-tagged p62 domains were in vitro transcribed/translated and labelled with [35S]-methionine. Recombinant GST and GST-FAT10 were bound to glutathione-Sepharose matrix and incubated with the in vitro translated [35S]-labelled HA-p62 constructs. The samples were boiled with a 10 % β-mercaptoethanol containing SDS-sample buffer and separated by SDS PAGE on 18 % gels. The HA-p62 constructs were detected by autoradiography and the GST fusion proteins were visualised by Coomassie Blue-staining. The monomeric wild-type HA-p62 protein has a molecular weight of 70 kDa, the HA-PB1 domain of 12,5 kDa, the HA-TRAF domain of 4,5 kDa, the N-terminal PEST domain as well as the HA-UBA domain of 6 kDa and the monomeric GST-FAT10 of 40 kDa. This experiment was only repeated once.
In the Coomassie Blue-staining, the recombinant GST (fig.31, lanes 2, 5, 8, 11, 14) and GST-FAT10 (fig.31, lanes 3, 6, 9, 12, 15) were detectable. In the autoradiogram, only the HA-p62 (fig.31, lane 1) and HA-p62(PB1 only) (fig.31, lane 4) were detectable. No signal was detectable for HA-p62(TRAF only), HA-p62(PEST only) or
Results
experiment however HA-p62 was pulled down with GST-FAT10 (fig.31, lane 3), but also with GST (fig.31, lane 2), indicating that it also binds to either the GSH-coupled agarose or to GST unspecifically.
Like in the transfection experiments (fig.30), also with the in vitro transcription and translation assay, only the wild-type HA-p62 and the isolated HA-PB1 domain were detectable (fig.31). Besides the start methionine, with the exception of the HA-p62(TRAF only) peptide, all isolated p62 domain peptides contain additional methionines and/or cysteines. Therefore, theoretically at least the HA-p62(PEST only) and HA-p62(UBA only) peptides should be easily detectable in the autoradiogram.