Presentation of endogenous and exogenous antigens is not affected by inactivation of E₁ ubiquitin-activating enzyme in temperature-sensitive cell lines

Little is known regarding the mechanism by which MHC class I-associated peptides are generated. Proteins can be targeted for degradation by the covalent attachment of ubiquitin. The first step in ubiquitin conjugation to proteins is its binding to E₁ ubiquitin-activating enzyme. To study the role of ubiquitin-targeted protein degradation in Ag processing, we used two mutant cell lines with temperature-sensitive E₁ proteins, and a recombinant vaccinia virus expressing wild-type human E₁. One of the cell lines examined (hamster ts20 cells) was previously reported to have a minimal capacity after a 1-h incubation at 41°C to present osmotically loaded OVA to a T cell hybridoma, as assessed by IL-2 release. Even after incubating the same cells for 1 h at 43°C, we failed to detect an E₁-related decrease in the presentation of biosynthesized or osmotically loaded OVA to splenic T cells, as measured by target cell lysis. We introduce the use of mouse tsA1S9 cells to Ag-processing studies and provide the initial biochemical characterization of their defect in protein ubiquitination. Relative to parental L929 cells, after thermal inactivation of E₁, these cells actually demonstrate enhanced presentation of endogenous or exogenous viral Ags to T cells. Our findings do not support a role for protein ubiquitination in Ag processing, and indicate that either the temperature-sensitive cell lines examined do not exhibit a sufficient reduction in ubiquitin-conjugating activity to affect the generation of antigenic peptides, or that ubiquitin-targeted proteolysis is not essential for processing the two exogenous and six endogenous Ags examined.