title: Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex.
abstract: The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.
authors.affiliation: From the Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 and.
authors.forename: Rafael
authors.initials: R
authors.lastname: Mattera
authors.affiliation: From the Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 and.
authors.forename: Carlos M
authors.initials: CM
authors.lastname: Guardia
authors.affiliation: The Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.affiliation: From the Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 and bonifacinoj@helix.nih.gov.
authors.forename: Juan S
authors.initials: JS
authors.lastname: Bonifacino
mesh_terms: D033964:Adaptor Protein Complex 4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D033964:Adaptor Protein Complex 4; D033942:Adaptor Proteins, Vesicular Transport; C000607390:tepsin protein, human
keywords: adaptor protein; clathrin; intracellular trafficking; membrane trafficking; neurodevelopment; peptide interaction; protein complex; protein motif; protein sorting; protein-protein interaction