Metazoans require the sorting nexin (SNX) protein, SNX27, to recycle hundreds of important transmembrane protein receptors from endosomes to the plasma membrane. Cargo recycling by SNX27 requires its interaction with retromer, a heterotrimer known to assemble on membranes with multiple sorting nexins, including SNX-BAR proteins and SNX3. SNX27 has also been functionally linked to SNX-BARs, but the molecular basis of this interaction has been unknown. We identify a direct biochemical interaction between the conserved and flexible SNX1/SNX2 N-terminus and full-length SNX27 using purified proteins in pulldown experiments. Sequence alignments indicate both SNX1 and SNX2 contain two short and conserved stretches of ... More
Metazoans require the sorting nexin (SNX) protein, SNX27, to recycle hundreds of important transmembrane protein receptors from endosomes to the plasma membrane. Cargo recycling by SNX27 requires its interaction with retromer, a heterotrimer known to assemble on membranes with multiple sorting nexins, including SNX-BAR proteins and SNX3. SNX27 has also been functionally linked to SNX-BARs, but the molecular basis of this interaction has been unknown. We identify a direct biochemical interaction between the conserved and flexible SNX1/SNX2 N-terminus and full-length SNX27 using purified proteins in pulldown experiments. Sequence alignments indicate both SNX1 and SNX2 contain two short and conserved stretches of acidic residues bearing a DxF motif in their flexible N-terminal regions. Biochemical pulldown and mapping experiments reveal forty residues in the N-terminus of either SNX1 or SNX2 can mediate binding to SNX27. SNX27 truncation analysis demonstrates the SNX27 FERM domain binds the SNX1 N-terminus. Calorimetry experiments quantified binding between the SNX1 N-terminus and SNX27 in the low micromolar affinity range (K ∼10 μM) and suggest the second DxF motif may play a more prominent role in binding. Mutation of either DxF sequence in SNX1 abrogates measurable binding to SNX27 in the calorimeter. Modelling from both predicted and experimentally determined structures suggests the SNX27 FERM domain could accommodate both DxF motifs simultaneously. Together, these data suggest SNX27 is directly linked to specific SNX-BAR proteins through binding acidic motifs in the SNX1 or SNX2 N-terminus.
