Adaptor protein (AP)-1/ó1B(-/-) mice have reduced synaptic-vesicle (SV) recycling and increased endosomes. Mutant mice have impaired spatial memory, and ó1B-deficient humans have a severe mental retardation. In order to define these ó1B(-/-) 'bulk' endosomes and to determine their functions in SV recycling, we developed a protocol to separate them from the majority of the neuronal endosomes. The ó1B(-/-) 'bulk' endosomes proved to be classic early endosomes with an increase in the phospholipid phosphatidylinositol 3-phosphate (PI-3-P), which recruits proteins mediating protein sorting out of early endosomes into different routes. ó1B deficiency induced alterations in the endosomal proteome reveals two major functions: SV protein storage and sorting into endolysosomes. Alternative endosomal recycling pathways are not up-regulated, but certain SV proteins are misrouted. Tetraspanins are enriched in ó1B(-/-) synaptosomes, but not in their endosomes or in their clathrin-coated-vesicles (CCVs), indicating AP-1/ó1B-dependent sorting. Synapses contain also more AP-2 CCV, although it is expected that they contain less due to reduced SV recycling. Coat composition of these AP-2 CCVs is altered, and thus, they represent a subpopulation of AP-2 CCVs. Association of calmodulin-dependent protein kinase (CaMK)-IIá, -ä and casein kinase (CK)-IIá with the endosome/SV pool is altered, as well as 14-3-3ç, indicating changes in specific signalling pathways regulating synaptic plasticity. The accumulation of early endosomes and endocytotic AP-2 CCV indicates the regulation of SV recycling via early endosomes by the interdependent regulation of AP-2-mediated endocytosis and AP-1/ó1B-mediated SV reformation.
14-3-3 Proteins Animals