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The tyrosine motifs of Lamp 1 and LAP determine their direct and indirect targetting to lysosomes

Institute for Biochemistry II, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany

View larger version (24K): [in a new window]   Fig. 1. Domain composition of LAP, Lamp 1, Limp II and their chimeras. The domains of Lamp 1 are represented by open bars, LAP domains by grey bars and Limp II domains by hatched bars. In a first set of chimeras, the lumenal and transmembrane domains of LAP were substituted with the respective domains from Lamp 1 or Limp II. In the second set of chimeras, the cytoplasmic tail of LAP was substituted for that of Lamp 1 or Limp II.

View larger version (74K): [in a new window]   Fig. 2. Detection of LAP at the plasma membrane by neuraminidase. Cells expressing LAP, LAP-Lamp-1 or LAP-Limp-II were metabolically labelled for 15 minutes and chased for one hour. Subsequently, the cells were incubated for one hour on ice with neuraminidase to allow desialylation of proteins present at the cell surface (lane B) or kept on ice without neuraminidase (lane A). An additional sample was incubated with neuraminidase for 20 minutes at 37°C (lane C). LAP was immunoprecipitated from cell extracts and subjected to isoelectric focusing. Desialylation by neuraminidase leads to a shift of LAP from acidic towards more basic pH (as indicated by the arrow). The numbers given below the lanes represent the fraction of desialylated LAP as a percentage to the total LAP.

View larger version (46K): [in a new window]   Fig. 3. Recycling of LAP and LAP chimeras from endosomes to the plasma membrane. Three dishes of cells expressing LAP were biotinylated on ice. Subsequently, the cells from the first dish (lane I) were harvested for determination of surface LAP. The remaining two dishes were incubated for 10 minutes at 37°C to allow endocytosis. Subsequently, the cells were put on ice and the biotin label present at the cell surface was removed using a reducing buffer system. After biotin stripping, the cells of the second dish (lane II) were collected for determination of internalised LAP, whereas the cells of the third dish were incubated a second time for 10 minutes at 37°C followed by biotin stripping for determination of recycled LAP. From each dish one-tenth was used to quantify the total amount of LAP (A), and the remaining nine-tenths were used to precipitate the biotinylated LAP using Streptavidin-Agarose (B). LAP was detected by western blotting. The numbers below the lanes give the fraction of biotinylated LAP as a percentage of total LAP (I), the fraction of internalised biotinylated LAP as a percentage of biotinylated LAP (II) and the fraction of recycled biotinylated LAP as a percentage of internalised biotinylated LAP (III). In addition, the same type of experiment is shown for cells expressing the LAP-Lamp-1 and LAP-Limp-II chimeras.

View larger version (28K): [in a new window]   Fig. 4. Cytoplasmic tail sequences of Limp II, Lamp 1, LAP and mutants derived from LAP or Lamp 1. The amino-acid sequences are shown using the one-letter code, with the C-terminus to the right. Signals known to be important for intracellular sorting are printed in bold. The colour of the bars corresponds to those used in Fig. 1 to indicate LAP (grey bars) or Lamp 1 (open bars) sequences. In LAP7, the C-terminal seven residues were removed by introducing a premature stop codon at the position of alanine 13. The following mutant proteins all contain the lumenal and the transmembrane part of LAP as a reporter. Their names indicate the type and origin of their tyrosine-sorting motif they contain: LAP7/YQTI is a mutant form of the LAP7 tail in which the LAP tyrosine motif was substituted for that of Lamp 1. In Lamp 1/YRHV, the tyrosine motif within the Lamp 1 tail sequence is substituted for that of LAP. LAP7/TI and Lamp 1/HV represent cytoplasmic tail mutations in which the last two residues of the tyrosine motifs of LAP7 and Lamp 1 were exchanged.

View larger version (29K): [in a new window]   Fig. 5. Binding of AP1 and AP2 to peptides representing the cytoplasmic tail of Lamp 1, LAP and LAP7. The peptides were immobilised on the surface of a biosensor and probed for the binding of AP1 or AP2 purified from pig brain. From the sensorgrams, the rate constants for association (ka), the dissociation (kd) and the equilibrium (KD) were calculated. It is notable that only the Lamp 1 peptide was able to bind to both adaptors, whereas LAP and LAP7 peptides were only able to bind to AP2.

View larger version (44K): [in a new window]   Fig. 6. Recycling between endosomes and the plasma membrane depends on the amino-acid composition of the tyrosine motif. LAP reporter proteins that contain either the LAP7 tail or the Lamp 1 tail, but both with variations in the position +2 and +3 of the tyrosine motif (LAP7/TI and Lamp 1/HV, Fig. 4), were expressed and assayed for recycling between endosomes and the plasma membrane exactly as described under Fig. 3. Changing the residues +2 and +3 within the LAP tyrosine motif to the respective residues of Lamp 1 (LAP7/TI) abolished recycling to a high extend, whereas changing the residues +2 and +3 within the Lamp-1 tyrosine motif to the respective LAP residues induces efficient recycling (Fig. 3; Table 2). Owing to the gel system used here, the mature form of LAP is visible in the total cell extracts (indicated by the asterisk).

View larger version (91K): [in a new window]   Fig. 7. The type of tyrosine motif determines the lysosomal delivery of LAP and Lamp 1. BHK cells expressing the same two LAP reporter proteins, which were described in Fig. 6, containing the two different variants of the tyrosine motifs of LAP and Lamp 1 (Fig. 1) were incubated for 15 minutes (A and B) or one hour (C and D) at 37°C in the presence of antibodies against the lumenal domain of LAP. Subsequently, the cells were fixed and stained for LAP (green colour in all images) and the early endosomal marker EEA1 (red colour in A and B) or endogenous Lamp 1 (red colour in C and D). Changing the residues +2 and +3 in the tyrosine motif of LAP to those of Lamp 1 (LAP7/TI) results in the efficient lysosomal delivery of the mutant within one hour (C) as indicated by the almost complete colocalisation with endogenous Lamp 1. Within 15 minutes of endocytosis, only a small fraction of the mutant can be detected in early endosomes (colocalization with EEA1 in A). Conversely, the substitution of the last two residues in the tyrosine motif of Lamp 1 for those of LAP results in endosomal retention of the mutant (colocalisation with EEA1 in B), which is also reflected by the small degree of colocalisation with endogenous Lamp 1 after one hour of endocytosis.

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