Abstract
The two known mannose 6-phosphate receptors (MPR46 and MPR300) both mediate the transport of Man-6-P-containing lysosomal proteins to lysosomes. However, the MPRs cannot be detected in lysosomes, instead they recycle between the plasma membrane and endosomes and between endosomes and the trans-Golgi network. Both, endocytosis from the plasma membrane and budding of transport vesicles from the trans-Golgi network involves the interaction of the receptor with the clathrin-coated vesicles-associated protein complexes AP1 and AP2. We have analyzed this interaction between the Golgi-restricted AP1 complex and the plasma membrane-restricted AP2 complex with the MPR46 tail in vitro by using a biosensor. AP1 and AP2 both bind to and dissociate from the MPR46 tail with similar kinetics. Using synthetic peptides corresponding to different MPR receptor tail regions in inhibition and binding studies, a common high affinity binding site for AP1 and AP2 and two separate high affinity binding sites for AP1 and AP2, respectively, were identified.
Highlights
Clathrin-coated vesicles (CCVs)1 are transport intermediates of vesicular traffic from the trans-Golgi network (TGN) and from the plasma membrane to endosomes
The AP1 complex has been shown to be restricted under normal conditions to the TGN, whereas the AP2 complex can only be detected in coated pits and CCVs derived from the plasma membrane
assembly proteins (APs) containing fractions show a specific binding to the immobilized MPR46 tail
Summary
Clathrin-coated vesicles (CCVs) are transport intermediates of vesicular traffic from the trans-Golgi network (TGN) and from the plasma membrane to endosomes. It is possible to induce relocation of adaptors to endosomal structures [2, 3] It is still not known how the AP complexes are recruited to their respective target membrane and released thereof, the complexes itself are well characterized biochemically and genetically (4 – 6). In vitro reconstitution assays and the use of proteolytically truncated AP complexes have revealed that the two b subunits mediate the interaction with clathrin [8]. For the interactions between adaptor complexes with different receptor tails, the necessity of a specific sequence motif involving a tyrosine (10 –12, 14, 15) or a di-leucine [16]2 was demonstrated. Recent studies using the yeast two-hybrid system have shown an interaction between tyrosine based sorting signals and the m-chains of AP1 and AP2 [17,18,19]
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