Although total UBA1 levels were unchanged, after oxidation for 60 min, we observed dramatic changes in the levels of BIAM-labeled UBA1 in both the membrane and cytosol fractions that suggested oxidative stress induces translocation of UBA1 from the cytosol to the membrane. Notably, in PrdxII(-/-) oxRBCs, ubiquitination levels were reduced about 75% in the membrane fraction after 90 min, even though UBA1 levels were increased. These results suggest ubiquitination levels are determined by UBA1 activity, not the level of UBA1 protein. Levels of ubiquitin conjugate (denoted ∼Ub) in HEK293T and CMT93 cells transfected with UBA1(C278S) or UBA1(C632S) were lower than in cells expressing UBA1(WT) or another cysteine mutant. During the reaction, UBA1(WT)∼Ub was nearly completely eliminated within 30 min, whereas UBA1(C278S)∼Ub and UBA1(C632S)∼Ub persisted. Within UBA1(C278S)∼Ub, the catalytic cysteine (Cys-632) remained intact; nonetheless, migration of UBA1(C278S)∼Ub and UBA1(C632S)∼Ub were similar. These data suggest that Cys-278 can affect Ub charging through a change in the structural conformation of UBA1, not through direct interaction at the UBA1-Ub interface.