The tyrosine-containing internalisation motif in the cytoplasmic domain of TGN38/41 lies within a nascent helix

TGN38 and TGN41 are isoforms of an integral membrane protein (TGN38/41) which is predominantly located in the trans-Golgi network of mammalian cells, but which constitutively recycles between the TGN and the plasma membrane. The cytoplasmic domain tetrapeptide sequence "YQRL" is responsible for the internalization of TGN38/41 from the plasma membrane. This sequence conforms to the tyrosine containing internalization motif ("YXXhydro," where X is any amino acid and hydro is any large bulky hydrophobic amino acid) found in other integral membrane proteins which are internalized from the plasma membrane via clathrin-coated vesicles. Structural predictions have suggested that the YXXhydro motif might adopt a tight turn structure in solution, a prediction supported previously by nuclear magnetic resonance (NMR) studies on short synthetic peptides corresponding to variants of the generic YXXhydro motif. We have synthesized a 21-amino acid peptide which encompasses the TGN38/41 internalization motif and used it as template for two-dimensional NMR analysis. The data from these experiments demonstrate that the internalization motif in the cytoplasmic domain of TGN38/41 lies within a nascent helix, not a tight turn. This is the first study to show that tyrosine containing internalization motifs do not necessarily adopt a beta-turn conformation.