Identification of a novel motif important for the activity of ring-type E3 ligases using MARCH1 as a model (100.52)

Abstract

Abstract The MARCH family of proteins are RING-CH type E3 ubiquitin ligases responsible for the ubiquitination and endocytosis of cell surface proteins related to immune response. We are particularty interested in MARCH1 and 8, two members of the family that are known to downregulate the cell surface expression of MHC II, CD86 , TfR and CD95. Canonical sorting motifs in the cytoplasmic portions of murine MARCH1 and human MARCH8 were identified and reported by others to be necessary for the function. We hypothesized that such motifs in human MARCH1 are important for it’s function. Methods: We used co-immunoprecipitations and FRET to monitor interaction of MARCH1 with various proteins. Protein expression, function and localization were determined by flow cytometry, confocal microscopy and western blotting. Results: Our results showed the presence of functional sorting motifs in both N- and C-terminal cytoplasmic portions of human MARCH1. However, these signals are not essential for the activity of MARCH1. We used a series of mutants to identify functional regions on MARCH1. Our results point to a novel motif. Interestingly, that motif is found in many other proteins and appears to be important for the function of other RING-type E3 ligases. Conclusions: Our results confirmed the presence of sorting motifs in MARCH1 and highlight important species-specific functional domains. The newly identified domain of MARCH1 will shed light on the structure of RING-type E3 ubiquitin ligases.