Phylogenetic and structural studies on the interaction of calcyon and clathrin assembly polypeptides/

Abstract

Calcyon is a member of a vertebrate specific gene family which includes Neuron Enriched Endosomal Protein of 21 kDa (NEEP21), and P19. Calcyon interacts with clathrin light chain, stimulates clathrin mediated endocytosis in brain, and regulates activity dependent excitatory synaptic transmission. Calcyon is required for internalization of excitatory neurotransmitter receptors and expression of long-term synaptic depression. Phylogenetic analyses (Muthusamy et a!. 2009) revealed a high degree of sequence homology between calcyon and the gene family members NEEP21 and P19. Like NEEP21 and calcyon, in situ hybridization in zebra fish shows that P19 is highly expressed in brain. Extensive database searches indicate that the gene family members could not be identified in invertebrates. Furthermore, calcyon orthologs were retrieved only from mammalian databases. Phylogenetic analyses led to the identification of sequence motifs recognized by the 1.1 subunit of Clathrin Assembly Polypeptides (AP) that are present only in calcyon and not in either NEEP21 or P19. Heterotetromeric complexes AP-1, AP-2, AP-3, and AP-4 regulate int~malization of cargo as well as the trafficking of cargo from the trans- Golgi network to endosomes, lysosomes, and related organelles. Studies show that AP-1 is involved in polarized trafficking of neuronal proteins. In neurons, while AP-2 is prominantly involved in synaptic vesicle endocytosis, AP-3 has been implicated in synaptic vesicle (SV) biogenesis, as well as in the compensatory endocytosis and targeting of SV proteins. AP 1.1 subunits interact with YXX0 type motifs and the cytoplasmic domain of calcyon contains two such tyrosine motifs. Yeast two-hybrid and GST pull down experiments show that calcyon directly interacts with AP-1, AP-2, and the ubiquitous as well as the neuronal isoforms of AP-3 in vitro and in brain. Pull down experiments with point mutations or C terminal truncations suggest that the second tyrosine motif of calcyon is essential for AP interaction. The relevance of these interactions was explored in vivo using mice harboring null-alleles of calcyon. Calcyon deletion in mice preferentially, yet not exclusively, altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 funtion. To test this hypothesis we focused on the hilar region of hippocampus, where levels of calcyon, AP-3 and AP-3 cargoes are abundant. We analyzed brain cryosections for the content of Zinc transporter 3 (ZnT3) and phosphatidylinositol-4-kinase type II alpha (PI4Klla), two well-defmed AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIa immunostaining is significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype associated with loss of AP-3. Taken together our data suggests that calcyon directly interacts with AP-3 via a YXX0 type motif, and preferentially regulates targeting of AP-3 cargoes destined to synaptic vesicles.