Abstract
Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals, but the invariant chain interacts only with AP1 and AP2, whereas LIMPII interacts strongly with AP3. In a series of in vitro experiments, we investigated the effect of residues upstream of the leucine pairs and demonstrated that these residues determine adapter binding, and certain residues favor interactions with AP3. Furthermore, constructs that interacted stronger with AP3 interacted weakly with AP1 and vice versa. Exchanging residues upstream of the leucine-based signal in LIMPII with those of the invariant chain reduced LIMPII binding to AP3 in vitro, and in vivo the corresponding LIMPII mutant was rerouted via the plasma membrane like the invariant chain. These preferential interactions of different leucine signals with different AP complexes may thus be the determining step sorting proteins from the trans-Golgi network to their final destinations. Proteins that interact with AP3 are sorted directly to endosomes/lysosomes, whereas proteins that interact with AP1 are sorted via a different route. At the same time, constructs that exhibited specificity for either AP1 or AP3 might still interact with AP2, suggesting that AP2 may recognize a wider variety of leucine signals. This is consistent with the suggested role of AP2 in internalization of proteins containing general leucine-based signals, including proteins that have been missorted to the plasma membrane.
Highlights
The adaptor protein complexes (APs)1 AP1, AP2, AP3, and AP4 are important components of the intracellular sorting machinery
We have previously demonstrated that AP1 and AP2 adaptor protein complexes could interact with the invariant chain in an in vitro assay monitored with surface plasmon resonance technique, and that such interactions were dependent on the intact leucine-sorting signals in the invariant chain [15]
We have shown that two other molecules that contain similar leucine signals, tyrosinase and lysosomal integral membrane protein II (LIMPII), only poorly interact with AP1 or AP2, but strongly bind to AP3 [15, 22]
Summary
The adaptor protein complexes (APs) AP1, AP2, AP3, and AP4 are important components of the intracellular sorting machinery They associate with transport vesicles along the secretory and endocytic pathways and can interact with membrane proteins that contain signals for sorting into the appro-. Each AP contains four polypeptides, called adaptins: two large chains of ϳ100 kDa, a medium chain of ϳ50 kDa, and a small chain of ϳ25 kDa. AP1 complexes are associated with transport clathrin-coated vesicles (CCVs) derived from the trans-Golgi network (TGN), AP2 complexes are associated with the endocytic CCVs, whereas the functional localization of AP3 and AP4 is not clear yet. Two molecules containing leucine-sorting signals are of particular interest for this study: the invariant chain (Ii) and lysosomal integral membrane protein II (LIMPII). LIMPII is a type III transmembrane protein, and has one leucine signal in its 20-amino acid cytoplasmic tail To corroborate our in vitro observations, we analyzed the intracellular fate of the wild type LIMPII and LIMPII with the invariant chain signal, transfected into MDCK cells
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