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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 [Trp.sup.19] [right arrow] Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either [Phe.sup.18] [right arrow] [Phe.sup.13] [right arrow] Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of [Trp.sup.19] for sorting of the CD-MPR in endosomes, with [Phe.sup.18], [Phe.sup.13], and several neighboring residues contributing to this function. The addition of the [Phe.sup.18][Trp.sup.19] 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 [Phe.sup.18] and [Trp.sup.19] 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.

Cysteine34 and Cysteine30 of the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor are reversibly palmitoylated in mouse L cells. Metabolic labeling and immunoprecipitation analysis reveals that binding of Cys34 to the cytoplasmic tail of the lipid bilayer is necessary for trafficking and lysosomal enzyme function. Mutation of Cysteine34 to Ala causes the receptor to accumulate in dense lysosomes and inhibits cathepsin D sorting ability in the Golgi. Rapid turning over of the palmitate occurs with a half life of nearly 2 hrs.

Alterations in the spacing of tyrosine-based signal YXXI of the cytoplasmic tail of lamp1 relative to the lipid bilayer inhibits the trafficking of membrane protein to lysosomes. Immunoelectron microscopy indicates that mutant forms of lamp1, Lamp d384 and Lamp S5 are present in lysosomes and contain high amount of endosomal structures compared to wild lamp1. A tyrosine based motif on lamp1 is necessary for YXXI motif mediated sorting of initial endosomes. Several different sorting sites are able to recognize the YXXI signal, and the way the signals are spaced in relation to the membrane may affect their functioning.

A signal present in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) restricts the receptor from trafficking to lysosomes. This preventative action is also supported by the transmembrane domain of the CD-MPR. A deterioration in the half-life of CD-MPR is noticed during the removal of the terminal 40 residues from the 67-amino acid cytoplasmic tail of the receptor.

Mutated cells of Saccharomyces cerevisiae were screened for defective 35S-alpha factor uptake as part of the determination of the proteins required for the internalization step in endocytosis. Two mutants, end3 and end4, were isolated which were not only defective in factor intake, but were temperature sensitive for growth and failed to accumulate lucifer yellow in the vacuoles formed. However, secretion and vacuole formation were normal. The end4 mutation specifically affected the steps of the endocytic pathway prior to the formation of endosomal compartments.

Insertion of bacteriophage coat proteins into the membrane of infected bacterial cells can be studied as a model system of protein translocation across membranes. The coat protein of the filamentous [...]