Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail

Abstract

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 --> Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 --> Ala or Phe13 --> Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.

Figure 1

Trp¹⁹ is essential for avoiding receptor trafficking to dense lysosomes. (A) Schematic illustration of the point mutants within the cytoplasmic tail of the CD-MPR. Amino acids replacing the wild-type sequence are shown in bold letters. (B) Mouse L cells stably expressing F13A, F18A, W19A, and F32A were preincubated with pepstatin A and leupeptin for 24 h. The cells were then homogenized with a ball bearing homogenizer, and postnuclear fractions were subjected to Percoll density gradient centrifugation (18% Percoll). The collected fractions were combined into pools I, II, and III (three each) and further analyzed by SDS/PAGE and immunoblotting with mAb 22D4. The upper band at ≈90 kDa is the dimeric form of the W19A receptor. The recovery of dimer varied with the different constructs. Both the monomeric and dimeric forms of the receptor were included in the quantitation summarized in Table 1.

Figure 2

FFWY→A, FFW→A, and FW→A accumulate in dense lysosomes. (A) Schematic illustration of the point mutants within the cytoplasmic tail of the CD-MPR. Amino acids replacing the wild-type sequence are shown in bold letters. (B) Mouse L cells stably expressing FFWY→A, FFW→A, and FW→A were preincubated with pepstatin A and leupeptin for 24 h and then fractionated as described in Fig. 1.

Figure 3

The Phe¹⁸-Trp¹⁹ sequence of the CD-MPR is sufficient to impair delivery of Lamp1 to lysosomes. (A) Schematic illustration of wild-type CD-MPR, wild-type Lamp1, and the chimeras LLM, LLL-M(FW), and LLL-M(AA). (B) Subcellular distribution of wild-type Lamp1, LLM, LLL-M(FW), and LLL-M(AA) on Percoll density gradients. Mouse L cells stably expressing wild-type Lamp1, LLM, LLL-M(FW), and LLL-M(AA) were preincubated with pepstatin A and leupeptin for 24 h and then fractionated as described in Fig. 1. Immunoblots of multiple gradients were quantitated, and the value of pool I (dense lysosomes) is expressed as a percentage of the sum of all three pools. The value for wild-type Lamp1 is from Reference 7. (C) Steady state cell surface distribution of wild-type Lamp1, LLL-M(FW), and LLL-M(AA). Cell surface proteins of mouse L cells stably expressing wild-type Lamp1, LLL-M(FW), and LLL-M(AA) were derivatized by using sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate. The cells were lysed, and the surface-biotinylated and internal Lamp1 molecules were immunoprecipitated by using a polyclonal anti-Lamp1 antibody (10). After solubilization of the first immunoprecipitate the samples were incubated with streptavidin–agarose beads to precipitate surface-biotinylated molecules. The proteins in the supernatant that did not bind to streptavidin (unbound, internal) and the proteins in the precipitate that did bind to streptavidin (bound, surface) were analyzed by SDS/PAGE and immunoblotting. Autoradiographs from multiple experiments were quantitated by scanning densitometry. The values presented in the graph represent the percentage of the different constructs detected at the cell surface (bound) relative to the total amount detected (bound and unbound combined).