Novel plant nuclear envelope-associated coiled-coil proteins

The nuclear envelope (NE) is a double lipid bilayer enclosing the eukaryotic genome. The metazoan nucleoskeleton includes the peripheral lamina and the internal nucleoskeleton. The lamina is composed of a network of intermediate filament (IF) proteins called lamins, as well as lamin- and/or chromatin-binding inner nuclear membrane (INM) proteins. The components of the metazoan lamina lack sequence homologues in plants. There is however evidence of a network of nuclear filamentous proteins underlying the NE. This study aims to characterise a novel family of NE-associated proteins (NEAP) in the model plant, Arabidopsis thaliana. The family consists of four proteins, AtNEAP1-4 conserved in plants restricted to the angiosperm clade. Their expression is ubiquitous with up-regulation in embryo, inflorescence and guard cells. NEAP protein structure consists of extensive coiled-coil (CC) domains, followed by a nuclear localisation signal (NLS) and a C-terminal transmembrane (TM) domain. Confocal microscopy shows that fluorescent protein tagged NEAP proteins localise to the nuclear periphery as part of highly immobilised stable complexes. Domain deletion mutants confirm the presence of functional NLS and TM domains, while their CC nature causes insolubility under high ionic salt and Triton X-100 conditions similar to other IF-like proteins. AtNEAP2 and AtNEAP3 interact with themselves as well as with AtNEAP1 and each other. NEAP proteins also interact with the classical and mid-SUN domain families. NEAP proteins also cause mis-localisation of the plant nuclear matrix constituent protein 1 from the nuclear periphery to the nucleoplasm. An A. thaliana cDNA library screen identified a basic leucine zipper transcription factor (TF), AtbZIP18 as a novel interactor of AtNEAP1. This is a first description of a chromatin-binding protein partner of the plant INM. Single and double NEAP knockout and knockdown mutants analysed displayed various defects in nuclear size, shape and positioning in different tissues. Therefore NEAP proteins appear to be involved in regulating nuclear morphology in plants. Thus as novel nuclear IF-like proteins that interact with a chromatin binding TF and have functions in regulating nuclear morphology, NEAP proteins are putative components of the plant lamina anchored at the INM