The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV.

Abstract

Lysosomal acid phosphatase (LAP) is rapidly internalized from the cell surface due to a tyrosine‐containing internalization signal in its 19 amino acid cytoplasmic tail. Measuring the internalization of a series of LAP cytoplasmic tail truncation and substitution mutants revealed that the N‐terminal 12 amino acids of the cytoplasmic tail are sufficient for rapid endocytosis and that the hexapeptide 411‐PGYRHV‐416 is the tyrosine‐containing internalization signal. Truncation and substitution mutants of amino acid residues following Val416 can prevent internalization even though these residues do not belong to the internalization signal. It was shown recently that part of the LAP cytoplasmic tail peptide corresponding to 410‐PPGY‐413 forms a well‐ordered beta turn structure in solution. Two‐dimensional NMR spectroscopy of two modified LAP tail peptides, in which the single tyrosine was substituted either by phenylalanine or by alanine, revealed that the tendency to form a beta turn is reduced by 25% in the phenylalanine‐containing peptide and by approximately 50% in the alanine‐containing mutant peptide. Our results suggest, that in the short cytoplasmic tail of LAP tyrosine is required for stabilization of the right turn and that the aromatic ring system of the tyrosine residue is a contact point to the putative cytoplasmic receptor.

Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor.

“…This result is similar to observations by Bansal and Gierasch and others on mutations in endocytosis signals that appear to destabilize the/3-turn, but differs in that the nascent helix is not also destabilized (Bansal and Gierasch, 1991;Backer et al, 1992;Lehmann et al, 1992). In these situations, one does not know if the mutation acts by altering the secondary structure and/or by changing a residue that is directly involved in recognition of the signal.…”

“…Comparison of NOESY spectra of peptides containing the endocytosis signal and mutated sequences for the LDLR, LAP, and the insulin receptor proved useful in correlating structure with function (Bansal and Gierasch, 1991;Ebede et al, 1991;Backer et al, 1992;Lehmann et al, 1992). As noted in the introduction, those studies correlated loss of a E-turn-like structure and function upon mutation.…”

“…Mutation of Tyr413 to Phe blocks endocytosis in MDCK cells, but not TGN to basolateral targeting, suggesting that the two types of signals can be separated (Prill et al, 1993). Analysis of the corresponding peptide by NMR indicates that this mutation destabilizes the /3-turn by about 25 % (Lehmann et al, 1992). Mutation of Tyr413 to Ala blocks both endocytosis and basolateral sorting, and NMR analysis shows that the/~-turn is destabilized by about 50% (Lehmarm et al, 1992).…”

Role of LAMP-2 in Lysosome Biogenesis and Autophagy

“…Filters were hybridized with a MPR46 and MPR300 cDNA probe (Kö ster et al, 1991) and a ␤-actin probe (CLONTECH, Palo Alto, CA). Hybridization and washing of the filters were performed as described (Lehmann et al, 1992).…”

Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation

“…Filters were hybridized with an NPC1 or NPC2 cDNA probe. Hybridzation and washing steps were performed as described (23). Hybridization with an actin cDNA probe was used as loading control.…”