Role of the ubiquitin-like modifier FAT10 in protein degradation and ...

Chapter 3
HDAC6 is required for the proper formation of FAT10-containing aggresomes
To assay whether HDAC6 is essential for the localization of FAT10 to the ag-
gresome, we set out to investigate the formation of aggresomes and subcellular
localization of FAT10 in cells derived from mice lacking the hdac6 gene (Zhang
et al., 2008b). Cells stably expressing an siRNA directed against HDAC6 have
previously been shown to be defective in the formation of polyubiquitin-containing
aggresomes. After inhibition of the proteasome, in comparison to wild-type cells,
fewer of these cells contained aggresomes and those aggresomes which did form
were smaller in size (Kawaguchi et al., 2003). Our experiments revealed HDAC6-
deficient cells to have a similar phenotype. When treated with 5 µM of the protea-
some inhibitor MG132 for 4 hours, HDAC6 knock-out cells were still able to form
polyubiquitin-containing aggresomes (Fig. 24B). However, fewer cells (approx-
imately 40% less) contained an aggresome in comparison to HDAC6 wild-type
cells (Fig. 25B) and the aggresomes which still formed were significantly smaller
in size (Fig. 25C). In those cells which were transiently transfected with HA-
FAT10, the subcellular localization – including the localization to aggresomes –
of FAT10 mirrored that of polyubiquitin (compare Figs. 24B h and p). The quan-
tification of aggresome size as determined by anti-HA immunofluorescence also
revealed HDAC6 knock-out cells to have significantly smaller FAT10-containing
aggresomes (Fig. 25D). As cells lacking HDAC6 are partially deficient in the for-
mation of FAT10- as well as polyubiquitin-containing aggresomes, HDAC6 cer-
tainly plays an important role in their formation, even though it does not appear
to be essential for the transport of neither polyubiquitin nor FAT10 to the aggre-
some or the formation of aggresomes in general.
Both ubiquitin-like domains of FAT10 interact with HDAC6 and localize to aggresomes
under proteasome inhibition
Since the isolated ubiquitin-like domains of FAT10 exhibit differences in their
ability to be degraded by the 26S proteasome and to interact with NEDD8 ul-
timate buster 1 long (Schmidtke et al., 2006), we decided to examine whether
there were also differences in their ability to bind to HDAC6 or to localize
to aggresomes. To investigate the interaction with HDAC6, we performed co-
immunoprecipitation experiments similar to the ones described above, only this
time we used wild-type HDAC6 and N-terminal fusions of either full length
FAT10 or the isolated N- and C-terminal ubiquitin-like domains of FAT10 with
GFP. We found that GFP-fusions with both the N- or C-terminal domain of FAT10
as well as full length FAT10 were able to interact with HDAC6 under proteasome
inhibition (Fig. 26A). In addition, immunofluorescence studies revealed that both
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