Abstract
The membrane-associated RING-CH (MARCH) family of membrane-bound E3 ubiquitin ligases regulates the levels of cell-surface membrane proteins, many of which are involved in immune responses. Although their role in ubiquitin-dependent endocytosis and degradation of cell-surface proteins is extensively documented, the features of MARCH proteins and their substrates that drive the molecular recognition events leading to ubiquitin transfer remain poorly defined. In this study, we sought to determine the features of human MARCH9 that are required for regulating the surface levels of its substrate proteins. Consistent with previous studies of other MARCH proteins, we found that susceptibility to MARCH9 activity is encoded in the transmembrane (TM) domains of its substrates. Accordingly, substitutions at specific residues and motifs within MARCH9's TM domains resulted in varying degrees of functional impairment. Most notably, a single serine-to-alanine substitution in the first of its two TM domains rendered MARCH9 completely unable to alter the surface levels of two different substrates: the major histocompatibility class I molecule HLA-A2 and the T-cell co-receptor CD4. Solution NMR analysis of a MARCH9 fragment encompassing the two TM domains and extracellular connecting loop revealed that the residues contributing most to MARCH9 activity are located in the α-helical portions of TM1 and TM2 that are closest to the extracellular face of the lipid bilayer. This observation defines a key region required for substrate regulation. In summary, our biochemical and structural findings demonstrate that specific sequences in the α-helical MARCH9 TM domains make crucial contributions to its ability to down-regulate its protein substrates.
Highlights
The membrane-associated RING-CH (MARCH) family of membrane-bound E3 ubiquitin ligases regulates the levels of cell-surface membrane proteins, many of which are involved in immune responses
In dendritic cells, when maturation is triggered by the uptake of antigenic cargo and/or exposure to Toll-like receptor ligands, MARCH1 activity is opposed by CD83 expression [10] and MARCH1 transcription stops [9, 11], resulting in major histocompatibility class II (MHC-II) and CD86 up-regulation at the cell surface and increased CD4ϩ T-cell stimulatory capacity
To quantify MARCH9-mediated down-regulation of target molecules from the cell surface, we designed a cell-based assay in which 293T cells endogenously expressing HLA-A2 were transduced with a lentiviral vector containing a doxycycline-inducible human MARCH9 expression cassette and mCherry expressed from a constitutive promoter
Summary
A serine in the first transmembrane domain of the human E3 ubiquitin ligase MARCH9 is critical for down-regulation of its protein substrates. MARCH9 has been shown to regulate ICAM-I [31] and a large number of B-cell surface proteins identified in a proteomic study of MARCH9-overexpressing cells [32] How this unusual family of membrane-bound E3 ligases recognizes this wide variety of substrate proteins and what, identifies a membrane protein for MIR/MARCH-mediated regulation remain open questions. Consistent with a possible role in direct interactions with substrate proteins, the TM domains of MIR and MARCH proteins contain many sequences that are commonly associated with specific TM–TM interfaces in membrane proteins [35] These include glycophorin A-like small amino acid motifs (glycine-XXX-glycine and derivatives containing alanine, serine, or threonine), strongly polar (glutamine and asparagine), polar/ aromatic (tyrosine and tryptophan), and ionizable (lysine, aspartic acid, glutamic acid, and histidine) residues that frequently drive TM interactions through close helix– helix packing, hydrogen-bonds, and strong ionic interactions (36 –44). An initial structural analysis using solution nuclear magnetic resonance (NMR) showed that these key residues are located in the ␣-helical portions of TM1 and TM2 that are closest to the extracellular face of the lipid bilayer, strongly implicating this particular region in MARCH9-mediated membrane protein regulation
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