Purpose: To understand the regulated degradation of KSHV K7.
Methods: Proteomic screen and immunofluoresence microscopy identified that K7 recruits polyubiquitin chains to membrane fractions; IP and GST pulldown verified the interaction between K7 and Iso T1; Protein stability assay and RQ-PCR demonstrated Iso T1 facilitates K7 degradation.
Results: The K7-containing membrane fraction contains a higher level of deubiquitinating (DUB) activity and K7 interacts with a cellular DUB, isopeptidase T1 (Iso T1). Mutational analyses revealed that the ubiquitin-associated domains of Iso T1 are necessary and sufficient to bind K7. Confocal microscopy and fractionation analyses indicated that K7 increases the membrane-associated Iso T1. Furthermore, the knockdown of IsoT1 by shRNA-mediated silencing greatly increased K7 ubiquitination even when proteasome activity was inhibited by lactacystin.
Conclusions: IsoT1 disassembles of free ubiquitin chains to facilitate K7 degradation.