Intra-membrane substrate recognition by membrane-tethered E3 ligases

© 2017 Dr Cyrus TanMembrane-associated RING-CH9 (MARCH9) is a member of a family of membrane-bound E3 ubiquitin ligases that down-regulates expression of cell-surface receptors and molecules via ubiquitin-mediated endocytosis. Members of the MARCH family have been implicated in regulating the immune response by controlling the surface levels of molecules responsible for antigen presentation and T cell stimulation. While substrate recognition has been shown to be driven by transmembrane (TM) domain interactions within the lipid bilayer for some family members, the molecular determinants of target recognition by MARCH9 remain undefined. This thesis describes parallel biochemical and biophysical approaches to determine the features of MARCH9 that mediate target recognition. Down-regulation of TM domain-swapped chimeras of substrate and non-substrate proteins by MARCH9 confirm that interactions within the membrane contribute to MARCH9 function. A mutagenesis analysis of the TM domain sequences of MARCH9 reveals specific sequence motifs that are critical for the down-regulation of two unrelated substrates (Chapter 3). Structural studies of the membrane-embedded region of MARCH9 by solution NMR produced a dynamic ensemble of backbone models that may reflect the inadequacy of micellar conditions used for studying this region in its native state (Chapter 4). To my knowledge, these results represent the most comprehensive analysis of the TM domains of MARCH9 to date, and provide a rationale for more detailed studies of the TM domains of the other MARCH proteins. Only a more complete understanding of MARCH-substrate interactions will allow us fully appreciate the complexity of substrate recognition by the MARCH proteins