Ubiquitin-conjugating enzyme E2 D1 (Ube2D1) mediates lysine-independent ubiquitination of the E3 ubiquitin ligase March-I

March-I is a membrane-bound E3 ubiquitin ligase belonging to the membrane-associated RING-CH (March) family. March-I ubiquitinates and down-regulates the expression of major histocompatibility complex (MHC) class II and cluster of differentiation 86 (CD86) in antigen-presenting cells. March-I expression is regulated both transcriptionally and posttranslationally, and it has been reported that March-I is ubiquitinated and that this ubiquitination contributes to March-I turnover. However, the molecular mechanism regulating March-I ubiquitination and the importance of March-I's E3 ligase activity for March-I ubiquitination are not fully understood. Here we confirmed that, although March-I is ubiquitinated, it is not ubiquitinated on a lysine residue, as a lysine-less March-I variant was ubiquitinated similarly as wildtype March-I. We found that March-I E3 ligase activity is not required for its ubiquitination and does not regulate March-I protein expression, suggesting that March-I does not undergo autoubiquitination. Knocking down ubiquitin-conjugating enzyme E2 D1 (Ube2D1) impaired March-I ubiquitination, increased March-I expression, and enhanced March-I-dependent down-regulation of MHC-II proteins. Taken together, our results suggest that March-I undergoes lysine-independent ubiquitination by an as yet unidentified E3 ubiquitin ligase that, together with Ube2D1, regulates March-I expression.