Peptides corresponding to the proposed coated pit internalization signal of the native low density lipoprotein receptor, NPVY, take up in aqueous solution a reverse-turn conformation with the Asn in position i and the fyr in position i+ 3. By contrast, peptides derived from receptors that are defective for endocytosis do not adopt the reverse turn. These internalization-defective receptors include those with a nonaromatic residue substituted for the Tyr and those with Asn+ Ala or Pro--Ala substitutions. While the tendency of an Asn-Pro sequence to induce a reverse turn was anticipated, the structural importance of an aromatic residue in position i+ 3 was not. The sequences associated with the internalization signal thus appear to play a critical conformational role that is required for endocytosis, probably by enabling binding to adaptor molecules. With the results presented in the accompanying paper on iysosomal acid phosphatase, we now have direct evidence for previous proposals of a general correlation of internalization signals with a turn conformational motif. introduction internalization of coated pit receptors requires information in their cytoplasmic tails (Lehrman et al., 1985; Mostov et al., 1988; Lobe1 et al., 1989; Miettinen et al., 1989; Jing et al., 1990; Thies et al., 1990). The recognition of receptors is believed to be mediated by binding of these cytoplasmic tail domains to the adaptins, components of the clathrincoated pit assembly (Pearse, 1988; Glickman et al., 1989). Among coated pit receptors, the low density lipoprotein (LDL) receptor, which carries plasmacholesterol into ceils, has been the subject of detailed studies of the sequence requirement for endocytosis. A genetic lesion (the JD mutation) in which 1 residue in the LDL receptor carboxyterminal cytopiasmic tail, Tyr-807, is substituted by Cys leads to an internalization defect and severe hypercholesterolemia (Brown and Goldstein, 1978). Mutagenesis and expression of the receptor in cultured cells demonstrated that any nonaromatic residue at position 807 caused an internalization block, while aromatic residues (Phe, Trp) functioned at near wild-type levels (Davis et al., 1988, 1987). From this result, it is clear that the native Tyr residue is not specifically required, either for chemical modification or for recognition. Additionally, mutagenesis of the receptor showed that a truncated tail, shortened from 50 to 22 residues, internalized at near wild-type levels (Davis et al., 1987). Further shortening caused reduced endocytosis of the receptor. In a recent study, Ala residues were introduced singly into the truncated 22 residue tail region of the LDL receptor at the 13 positions that are the most highly conserved among LDL receptors from different species (Chen et al., 1990). The Ala substitutions at four positions, F802, N804, P805, and Y807, caused substantially decreased levels of internalization, and only minor effects were elicited at the other positions. From this result and comparisons among different coated pit receptors, Chen et al.(1990) proposed that the sequence NPXY (where X is any amino acid) constitutes a signal for internalization. How this internalization sequence may mediate endocytosis is not clear. The residues present in the internalization signal may favor a specific conformation to present a recognition domain to adaptin. Alternatively, the conformational information may lie elsewhere in the tail sequence, and the NPXY sequence may provide functional groups necessary for recognition. A recent proposal suggests that internalization signals take up a reverse turn (Collawn et al., 1990). The authors searched known protein structures for sequences similar to those implicated in …