No abstract

In eukaryotic cells, complex regulatory mechanisms involving numerous proteins must operate to ensure the temporal and spatial specificity of intracellular membrane-trafficking pathways. We and others have identified three members of the protein kinase C and casein kinase 2 substrate in neurons (PACSIN) protein family, also named syndapin and focal adhesion protein 52 (FAP52), which participate in rearrangements of actin networks during endocytosis [1][2][3][4][5]. In contrast to the neuron-specific PACSIN 1, other members of the PACSIN protein family show a broader tissue distribution [2,4,6]. Via their C-terminal Src homology 3 (SH3) domains, PACSIN proteins bind to proline-rich domains of dynamin, synapsin and synaptojanin, three proteins also involved in vesicle endocytosis, as well as to neural Wiskott-Aldrich syndrome protein The ability of protein kinase C and casein kinase 2 substrate in neurons (PACSIN) ⁄ syndapin proteins to self-polymerize is crucial for the simultaneous interactions with more than one Src homology 3 domain-binding partner or with lipid membranes. The assembly of this network has profound effects on the neural Wiskott-Aldrich syndrome protein-mediated attachment of the actin polymerization machinery to vesicle membranes as well as on the movement of the corresponding vesicles. Also, the sensing of vesicle membranes and ⁄ or the induction of membrane curvature are more easily facilitated in the presence of larger PACSIN complexes. The N-terminal Fes-CIP homology and Bin-Amphiphysin-Rvs (F-BAR) domains of several PACSIN-related proteins have been shown to mediate self-interactions, whereas studies using deletion mutants derived from closely related proteins led to the view that oligomerization depends on the formation of a trimeric complex via a coiled-coil region present in these molecules. To address whether the model of trimeric complex formation is applicable to PACSIN 1, the protein was recombinantly expressed and tested in four different assays for homologous interactions. The results showed that PACSIN 1 forms tetramers of about 240 kDa, with the self-interaction having a K D of 6.4 · 10 )8 m. Ultrastructural analysis of these oligomers after negative staining showed that laterally arranged PACSIN molecules bind to each other via a large globular domain and form a barrel-like structure. Together, these results demonstrate that the N-terminal F-BAR domain of PACSIN 1 forms the contact site for a tetrameric structure, which is able to simultanously interact with multiple Src homology 3 binding partners.