Abstract
The AP1/σ1B knockout causes impaired synaptic vesicle recycling and enhanced protein sorting into endosomes, leading to severe intellectual disability. These disturbances in synaptic protein sorting induce as a secondary phenotype the upregulation of AP2 CCV mediated endocytosis. Synapses contain canonical AP2 CCV and AP2 CCV with a more stable coat and thus extended life time. In AP1/σ1B knockout synapses, pool sizes of both CCV classes are doubled. Additionally, stable CCV of the knockout are more stabilised than stable wt CCV. One mechanism responsible for enhanced CCV stabilisation is the reduction of synaptojanin1 CCV levels, the PI-4,5-P2 phosphatase essential for AP2 membrane dissociation. To identify mechanisms regulating synaptojanin1 recruitment, we compared synaptojanin1 CCV protein interactome levels and CCV protein interactions between both CCV classes from wt and knockout mice. We show that ITSN1 determines synaptojanin1 CCV levels. Sgip1/AP2 excess hinders synaptojanin1 binding to ITSN1, further lowering its levels. ITSN1 levels are determined by Eps15, not Eps15L1. In addition, the data reveal that reduced amounts of pacsin1 can be counter balanced by its enhanced activation. These data exemplify the complexity of CCV life cycle regulation and indicate how cargo proteins determine the life cycle of their CCV.
Highlights
Synapses contain two classes of AP2 CCV
Compared to wt stable CCV, stCCV have less of the clathrin disassembly protein Hsc[70], their AP2 complexes are hyperactivated for stable membrane binding and they have less of the PI-4,5-P2 phosphatase synaptojanin[1]
If endophilin is recruited via its SH3-domain by CCV proteins, its BAR-domain can recruit the Arf6-GEF EFA661 (Fig. 2A)
Summary
Synapses contain two classes of AP2 CCV (adaptor-protein complex 2; clathrin-coated-vesicles). Git[1] transport via stCCV points to important functions of stCCV in the regulation of AZ plasticity[1] These changes in AP2 CCV pathways are a secondary phenotype of the AP1/σ1B ko. We identified three molecular mechanisms responsible for the enhanced stability of AP2 stCCV1 They affect all layers of a CCV: the outer clathrin basket, the inner AP2 layer and the membrane phospholipid composition. Compared to wt stable CCV, stCCV have less of the clathrin disassembly protein Hsc[70], their AP2 complexes are hyperactivated for stable membrane binding and they have less of the PI-4,5-P2 phosphatase synaptojanin[1]. Understanding the mechanisms regulating synaptojanin[1] activities is important for CCV life cycle regulation, but is essential to understand the development of several severe neurological diseases. Synaptojanin[1] deficiencies induce strong phenotypes in SV recycling, whereas autophagosomes are not abundant in mature synapses[32,33]
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