Characterisation of Enaptin and Sun1, two novel mammalian nuclear envelope proteins

The nucleus is a highly dynamic organelle showing migratory events during various developmental and cellular processes. These are made possible by its association with the microtubule network and, as shown recently, with the microfilament network. We have characterised Enaptin, a giant protein located at the nuclear membrane, which may provide a link to the actin cytoskeleton. Enaptin has an actin binding domain at the N-terminus with which it binds to the actin cytoskeleton and a transmembrane domain at the C-terminus, which tethers it to the nuclear envelope (NE). Western blotting experiments with antibodies specific to the ABD of Enaptin detect a 400 kDa protein in various cell lines and a 165 kDa protein in mouse brain tissue. We concentrated on the properties of the very C-terminus composed of the last 30 amino acids after the transmembrane domain of Enaptin. A similar stretch of amino acids is found in the related NUANCE and is also highly conserved in proteins of the NE across different species from C. elegans to humans. We show that the conserved amino acids in Enaptin and NUANCE bind to the C-terminus of Sun1 in yeast two hybrid and GST pull down experiments. Sun1 is a novel NE protein with three transmembrane domains in the middle. The C-terminal end of Sun1 is homologous to the SUN domain, which derives its name from its homology between Sad1 (S. pombe protein associated with the spindle pole body) (Iain and Mitsuhiro, 1995) and UNC-84 (C. elegans orthologue of Sun1) (Malone et al., 1999). The SUN domain of Sun1 is however not engaged in the binding of Enaptin. Instead, the interaction site is located in a region neighboring the SUN domain. A dominant negative version of Sun1 (GFP-CT+3TM), which displaces endogenous Sun1 from the NE also displaces NUANCE and emerin, a protein located in the inner nuclear membrane, underlining the fact that NUANCE localisation to the NE is also Sun1 dependent. Further experiments were directed at the identification of the site of location of Sun1 employing expression of GFP-tagged Sun1 as well as antibodies generated against the N- and C-terminus of Sun1. Immunofluorescence studies performed after digitonin permeabilisation finally located the protein at the inner nuclear membrane. In further experiments we analysed the requirements for the nuclear envelope localisation of Sun1. The results point to interactions with different proteins of the NE and also to the importance of the coiled coil domain.