|dc.description.abstracteng||In contrast to the well-studied process of nucleocytoplasmic transport of soluble macromolecules, little is known about the targeting of integral membrane proteins to the inner nuclear membrane (INM), therefore the targeting of INM-proteins was studied in this work.
Two major models haven been proposed for INM-protein transport, the diffusion-retention model, which assumes passive diffusion of proteins from the ER membrane, across the nuclear pore complex (NPC) to the INM and the transport factor-mediated model, where membrane proteins are believed to translocate across the NPC into the nucleus upon binding to nuclear import receptors. In this work, the targeting of proteins to the INM was studied for several tail-anchored (TA)-proteins, which contain only a single C-terminal transmembrane domain (TMD). As a model protein, the well-established INM-protein emerin was used. With the help of membrane insertion assays, we showed that emerin, expressed in bacteria or a cell-free lysate, can be post-translationally integrated into ER membranes by the TRC40-pathway.
The involvement of the TRC40-system was shown by use of inhibitory fragments of the ER-membrane receptors WRB and CAML in the membrane insertion reaction. An interaction between emerin and TRC40 could further be confirmed by proximity ligation assays. Additionally, TRC40 knockdown experiments demonstrated a reduction of emerin localized at the nuclear envelope.
An important objective of this work was to verify correct targeting of emerin and other integral membrane proteins to the INM. To accomplish this, we performed differential permeabilization experiments, and established a rapamycin-based dimerization assay. These experiments allowed us to confirm the localization of emerin at the INM in a microscopy based approach.
In addition to wild type emerin, we also analyzed mutants. Mutations in the emerin gene in the TMD or in the C-terminal part lead to the neuromuscular disease Emery-Dreifuss muscular dystrophy. As the TMD of emerin is crucial for anchoring of emerin in a membrane, several C-terminal emerin mutations were selected and studied for their post-translational membrane insertion and localization at the INM. Several of these mutants showed severely disturbed TRC40-binding, membrane integration and INM-targeting.
The efficiency of targeting to the INM might therefore rely on properties within the TMD or at least the C-terminal region of emerin. The disease could therefore be a consequence of an impaired transport process or a reduced retention at the INM.
Apart from emerin, other putative tail-anchored INM-proteins like LAP2β, LEMD1, PTP1B, VAPB and LRRC59 were confirmed to localize to the INM using the rapamycin assay. Furthermore, some of them were shown to use a post-translational membrane insertion mechanism at the ER.||de