Functions and molecular mechanisms of AP-1 and AP-2 complexes and their subunits

• Applicant: Professor Dr. Peter V. Schu
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2008 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 86873670

Vesicular protein sorting in late, post trans-Golgi network (TGN) secretory and endocytic routes is mediated largely by the family of heterotetrameric adaptor-protein complexes AP-1 to AP-5. Deficiency in AP-1 or in AP-2 is embryonic lethal. Deficiency in one of the other three complexes causes neurological diseases. AP-1 and AP-2 form ,Clathrin-coated-Vesicles‘ (CCV). AP-2 exclusively at the plasma membrane, where it mediates endocytosis. Our mouse ,knock-out‘ models of AP-1 adaptins demonstrated functions in bidirectional TGN/endosom transport. AP-1 differs from the other AP-complexes by its numerous tissue-specific isoforms. The γ1/AP-1 complex is formed by γ1, β1, μ1A and σ1A adaptins. These are ubiquitous and essential for vertebrate embryogenesis. Isoforms σ1B and σ1C have a complementary tissue-specific expression and tissues express AP-1/σ1A and AP-1/σ1B or AP-1/σ1C. The γ2 isoform has not been studied intensively. γ1 is found in all eukaryotes, γ2 only in vertebrates/plants. We studied σ1B-adaptin function in a mouse ,knock-out‘. σ1B-deficiency does not impair vitality, but causes severe deficiencies in learning and memory. Its gene is part of the X-chromosome in mouse and man. We demonstrated impaired in hippocampal synaptic vesicle recycling, which is slowed down and incomplete. In parallel endosomes are enlarged. Thus AP-1 is essential for the recycling of synaptic vesicles via endosomal intermediates. The ubiquitous γ1/σ1A AP-1 complex accumulates on these endosomes and stimulates their maturation into late, multivesicular endosomes via recruiting ArfGAP1/Rabex-5. Rabex-5 activates the Rab5/Vps34 kinase pathway, which synthesises PI3P, essential for the maturation. γ1/σ1B AP-1 inhibits this pathway by competing for Rabex-5 binding and preventing formation of the stable γ1/σ1A AP-1/ArfGAP1/Rabex-5 complex. Surprisingly, endocytic AP-2 CCV accumulate in σ1B knockout synapses. This is surprising, because with the recycling of synaptic vesicles the most active synaptic vesicular transport route is impaired. We purified these synaptic CCV and characterised them biochemically to unravel the molecular mechanisms leading to their accumulation. σ1B is part of γ1 und γ2 AP-1 complexes and we studied γ2 functions to discriminate them from γ1 functions. Also γ2 is ubiquitous, but its expression levels do not correlate with the activities of secretory pathways of a tissue, as do the expression levels of γ1. γ2 is, like γ1, essential for embryogenesis. Also γ2 forms AP-1 complexes, like γ1, and it also binds to the TGN and forms CCV. γ2/σ1B AP-1 inhibits γ2/σ1A AP-1 functions. Thus γ1 and γ2 AP-1 fulfil different essential functions in development. We want to study the mechanisms regulating these AP-1 and AP-2 mediated protein sorting and transport routes. We identified kinases and phosphorylation sites in proteins specifically altered by the σ1B-deficiency. AP-1 also binds E3-ubiquitin ligases and we want to study their functions in synapses.

• DFG Programme: Research Grants