The interaction of assembly protein AP180 and clathrin is inhibited by multi-site phospho-mimetics.

Clathrin-mediated endocytosis at the nerve terminal is dependent on assembly protein 180 (AP180) and adapter protein complex 2 (AP2). Both membrane adapter proteins bind to each other and to clathrin, to drive assembly of the clathrin coat over nascent synaptic vesicles. Using knowledge of in vivo phosphorylation sites, AP180 was mutated to determine the effect on binding. N-terminally truncated AP180 exhibited phospho-mimetic (Ser/Thr to Glu)-dependent interaction with AP2, but not clathrin. C-terminally truncated and full length phospho-mutant AP180 bound less AP2 than wild type. However, there was no difference in AP2 binding for the phospho-mimetic or phospho-deficient (Ser/Thr to Ala) AP180 mutants. Thus, the phospho-mutant approach did not provide clarity for the role of phosphorylation in AP180-AP2 binding. Clathrin exhibited a phospho-mimetic-dependent interaction with full-length AP180. Furthermore, phospho-mimetic AP180 was deficient at assembling clathrin cages. These latter discoveries support a model where AP180 phosphorylation inhibits clathrin binding and assembly.

The ∼ 16 kDa C-terminal sequence of clathrin assembly protein AP180 is essential for efficient clathrin binding.

Brain-specific AP180 is present in clathrin coats at equal concentration to the adapter complex, AP2, and assembles clathrin faster than any other protein in vitro. Both AP180 and its ubiquitously expressed homolog clathrin assembly lymphoid myeloid leukemia protein (CALM) control vesicle size and shape in clathrin mediated endocytosis. The clathrin assembly role of AP180 is mediated by a long disordered C-terminal assembly domain. Within this assembly domain, a central acidic clathrin and adapter binding (CLAP) sub-domain contains all of the known short binding motifs for clathrin and AP2. The role of the remaining ∼ 16 kDa C-terminal sequence has not been clear. We show that this sequence has a separate function in ensuring efficient binding of clathrin, based on in vitro binding and ex vivo transferrin uptake assays. Sequence alignment suggests the C-terminal sub-domain is conserved in CALM.

The Biochemical Properties and Functions of CALM and AP180 in Clathrin Mediated Endocytosis.

Clathrin-mediated endocytosis (CME) is a fundamental process for the regulated internalization of transmembrane cargo and ligands via the formation of vesicles using a clathrin coat. A vesicle coat is initially created at the plasma membrane by clathrin assembly into a lattice, while a specific cargo sorting process selects and concentrates proteins for inclusion in the new vesicle. Vesicles formed via CME traffic to different parts of the cell and fuse with target membranes to deliver cargo. Both clathrin assembly and cargo sorting functions are features of the two gene family consisting of assembly protein 180 kDa (AP180) and clathrin assembly lymphoid myeloid leukemia protein (CALM). In this review, we compare the primary structure and domain organization of CALM and AP180 and relate these properties to known functions and roles in CME and disease.

Differential phosphorylation of dynamin I isoforms in subcellular compartments demonstrates the hidden complexity of phosphoproteomes.

Large-scale comparative phosphoproteomics studies have frequently been done on whole cells or organs by conventional bottom-up mass spectrometry approaches, that is, at the phosphopeptide level. Using this approach, there is no way to know which protein isoforms the phosphopeptide signal originated from. Also, as a consequence of the scale of these studies, important information on the localization of phosphorylation sites in subcellular compartments is not surveyed. As a case study, we investigated whether the isoforms of dynamin I (dynI), at the whole brain and subcellular level, had differential phosphorylation. We first established that the dynI isoforms xa, xb, and xd were expressed in nerve terminals. Our investigation revealed that dynI xa was constitutively phosphorylated to a higher extent than the other isoforms despite identical sequences in the phosphorylated subdomains. DynI xa had a 10-fold higher stoichiometry of diphosphorylation at Ser-774 and Ser-778 than dynI xb and xd combined. Diphosphorylation was 2-fold enriched in nerve terminals relative to whole brain and was preferentially targeted for stimulus-dependent dephosphorylation. Phospho-Ser-851 and Ser-857 were depleted from nerve terminals. Our data reveals major differential phosphorylation of dynI phosphosites in different variants and in different neuronal compartments that would be completely imperceptible to a large-scale phosphoproteomics approach.