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First published online 25 November 2008
doi: 10.1242/jcs.035006

Journal of Cell Science 121, 4079-4088 (2008)
Published by The Company of Biologists 2008
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The ubiquitin-like modifier FAT10 interacts with HDAC6 and localizes to aggresomes under proteasome inhibition

Birte Kalveram, Gunter Schmidtke and Marcus Groettrup*

Department of Biology, Division of Immunology, University of Constance, Universitätsstrasse 10, 78457 Konstanz, Germany


Figure 1
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  		Fig. 1. HDAC6 interacts with FAT10 only under proteasome inhibition and has no influence on the degradation rate of FAT10. (A) HEK293T cells, transiently transfected with HA-FAT10 and either wild-type (wt) FLAG-HDAC6, a {Delta}BUZ mutant or empty vector (–), were treated with 5 µM of the proteasome inhibitor MG132, or DMSO as a negative control, for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (B) HEK293T cells were transiently transfected with FLAG-HDAC6 followed by treatment with 500 U/ml TNF-{alpha} and 200 U/ml IFN-{gamma} for 16 hours, and 5 µM MG132 for another 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (C) HEK293T cells transfected with either HA-FAT10 alone (left panel), HA-FAT10 and FLAG-HDAC6 together (center panel) or FLAG-HDAC6 alone (right panel) were pulse-labeled with [35S]-methionine and chased for the indicated times (hours). Cell lysates were subjected to anti-HA and anti-FLAG immunoprecipitation followed by SDS-PAGE and autoradiography.

 

Figure 2
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  		Fig. 2. Both the BUZ domain and the first catalytic domain (CAT1) of HDAC6 are able to bind FAT10. (A) HEK293T cells, transfected with HA-FAT10 and the indicated FLAG-HDAC6 mutants, were treated with 5 µM MG132, or DMSO as a negative control, for 6 hours. Cell lysates were subjected to anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). Asterisks denote the position of antibody light chains. (B) Schematic representation of the mutants used in A. CAT1, first catalytic domain; CAT2, second catalytic domain; BUZ, ubiquitin-binding zinc finger.

 

Figure 3
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  		Fig. 3. Both full-length HDAC6 and the isolated CAT1 and BUZ domains interact with FAT10 in vitro. The diglycine motif of FAT10 is not required for its interaction with HDAC6. (A) GST-pulldown of [35S]-methionine-labeled in vitro transcribed and translated HDAC6 mutants with recombinant FAT10. Bound proteins were analyzed by SDS-PAGE and autoradiography. wt, wild type; CAT1, first catalytic domain; BUZ, ubiquitin-binding zinc finger. (B) Coomassie-staining of the recombinant proteins used in A. (C) GST-pulldown of [35S]-methionine-labeled HDAC6 with recombinant wild-type FAT10 or FAT10 lacking the C-terminal diglycine motif ({Delta}GG). (D) Coomassie-staining of recombinant FAT10 used in C.

 

Figure 4
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  		Fig. 4. Neither acetylation of FAT10 nor catalytic activity of HDAC6 are required for interaction between the two proteins. (A) HEK293T cells transfected with HA-FAT10 and either wild-type (wt) FLAG-HDAC6 or a catalytically dead mutant (mut) were treated with 5 µM MG132, or DMSO as a negative control, for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (B) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5 µM MG132, 1 µM nocodazole, 5 µM of the HDAC inhibitor trichostatin A (TSA) or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (C) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5 µM MG132, 20 µM tubacin or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (D) HEK293T cells, transfected with FLAG-HDAC6 and either wild-type (wt) or a lysine-less HA-FAT10 mutant (K0), were treated with 5 µM MG132 or DMSO for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (E) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5 µM MG132, 5 µM TSA or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB) with an acetyl-lysine antibody (AcK). The asterisk denotes the position of the antibody light chain; the arrowhead denotes the position of HA-FAT10 on the anti-AcK western blot.

 

Figure 5
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  		Fig. 5. FAT10 localizes to aggresomes under proteasome inhibition. (A) HEK293T cells transiently transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5 µM MG132, or DMSO as a negative control, for 6 hours and immunostained with antibodies for HA (green), FLAG (red) and DAPI (blue; a-h). HEK293T cells transfected with HA-FAT10 were treated with 5 µM MG132 or DMSO for 6 hours and immunostained with anti-HA (green), DAPI (blue) and either anti-{gamma}-tubulin (red; i-p), anti-ubiquitin (red; q-t) or anti-20S proteasome (red; u-x). (B) HEK293T cells treated with TNF-{alpha} and IFN-{gamma} for 16 hours were incubated in the presence of 5 µM MG132 or DMSO for an additional 6 hours and immunostained with antibodies against endogenous FAT10 (green), ubiquitin (red) and DAPI (blue). Scale bars: 5 µm.

 

Figure 6
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  		Fig. 6. The model conjugate FAT10-GFP localizes to aggresomes under proteasome inhibition. (A) HEK293T cells transfected with FLAG-HDAC6, GFP or FAT10-GFP were treated with 5 µM MG132 or DMSO for 6 hours and immunostained with anti-GFP antibody (green), anti-FLAG antibody (red) and DAPI (blue). Scale bars: 5 µm. (B) Quantification of the percentage of cells with GFP-containing aggresomes, n≥100. Error bars represent the standard error of the mean (s.e.m.); statistical analysis was carried out using Student's t-test.

 

Figure 7
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  		Fig. 7. Localization of FAT10 to aggresomes is dependent on the tubulin network and on HDAC6. (A) HEK293T cells transfected with HA-FAT10 were treated with 5 µM MG132, 1 µM nocodazole or mock treated for 6 hours, and immunostained with anti-HA antibody (green) and DAPI (blue). (B) HDAC6 wild-type (a-h) or knock-out (i-p) cells were transiently transfected with HA-FAT10 followed by treatment with 5 µM MG132 or DMSO for 4 hours and immunostained with anti-HA antibody (green), anti-ubiquitin antibody (red) and DAPI (blue). Scale bars: 5 µm.

 

Figure 8
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  		Fig. 8. HDAC6-deficient cells have defects in aggresome formation. (A) Verification of HDAC6 knockout by western blot. (B) Quantification of the percentage of cells with ubiquitin-containing aggresomes, n≥100. (C) Quantitative evaluation of the size of ubiquitin-containing aggresomes, n≥50. (D) Quantitative evaluation of the size of FAT10-containing aggresomes, n≥10. Error bars represent the standard error of the mean (s.e.m.); statistical analysis was carried out using Student's t-test.

 

Figure 9
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  		Fig. 9. Both of the ubiquitin-like domains of FAT10 interact with HDAC6 and localize to aggresomes under proteasome inhibition. (A) HEK293T cells transfected with FLAG-HDAC6 and the indicated GFP fusions were treated with 5 µM MG132 for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). FAT10-N-GFP, N-terminal domain of FAT10 fused to GFP; FAT10-C-GFP, C-terminal domain of FAT10 fused to GFP. (B) HEK293T cells transfected with either the N-terminal (a-c) or C-terminal (d-f) domain of FAT10 fused to GFP were treated with 5 µM MG132 for 6 hours and immunostained with anti-GFP antibody (green) and DAPI (blue). Scale bars: 5 µm.

 

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© The Company of Biologists Ltd 2008