Jun Xiao1, Hui Wu, Lingzhi Peng, Mengdie Chi, Hao Feng. 1. Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education of China, College of Life Science, Hunan Normal University, Changsha, China, 410081.
Abstract
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.
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.
Authors: J J Russo; R A Bohenzky; M C Chien; J Chen; M Yan; D Maddalena; J P Parry; D Peruzzi; I S Edelman; Y Chang; P S Moore Journal: Proc Natl Acad Sci U S A Date: 1996-12-10 Impact factor: 11.205