The ISG15 conjugation system

Abstract

ISG15 is a ubiquitin-like modifier that is expressed in response to type 1 interferon signaling (IFN-α/β) and plays a role in antiviral responses. The core E1, E2, and E3 enzymes for ISG15 are Ube1L, UbcH8, and Herc5, respectively, and these are all also induced at the transcriptional level by IFN-α/β. We recently showed that Herc5 associates with polysomes and modifies target proteins in a cotranslational manner. Here, we describe the expression of the core conjugating enzymes in human cells, the detection of ISG15 conjugates, and the methods for fractionation of Herc5 with polysomes.

Fig. 1.

Co-transfection of ISG15 with mutant forms of the ISG15 conjugating enzymes. HEK293T cells were transfected with plasmids expressing the indicated wild-type or mutant proteins. The plasmid expressing ISG15 contains an N-terminal 3×FLAG tag and the plasmid expressing Herc5 contains an N-terminal TAP tag. Cell extracts were prepared 24 h posttransfection and analyzed by immunoblotting with an antibody against the FLAG tag.