Analysis of Protein Transport to the Inner Nuclear Membrane

The eukaryotic cell is divided into different compartments. The biggest compartment is the nucleus which is formed by one lipid bilayer folded into two membranes: the outer nuclear membrane (ONM) and the inner nuclear membrane (INM). The ONM is continuous with the endoplasmic reticulum (ER) membrane and merges with the INM at a curved piece of membrane at the level of the nuclear pore complex (NPC). The NPC controls the exchange of proteins or other molecules between the cytoplasm and the nuclear compartment. While the active transport of soluble proteins via the NPC mediated by transport factors is well studied, less is known about the targeting of membrane proteins to the INM. Most membrane proteins are first inserted into the ER-membrane before they are targeted to their destination. A nascent polypeptide chain can be inserted into the ER-membrane during translation in a co-translational manner or post-translationally after termination of translation. The best described route of post-translational insertion into the ER-membrane of tail-anchored proteins, which are membrane proteins with a very short C-terminus, is the TRC (transmembrane domain-recognition complex) pathway. In this study, the insertion mechanism of the single-pass membrane protein LRRC59 (leucine-rich repeat-containing protein 59) into the ER-membrane is analyzed by in vitro microsome integration assays. LRRC59 is shown to be a tail-anchored protein, which can be post-translationally inserted independently of the TRC pathway. In former studies, LRRC59 was found at the ER-membrane and the nuclear envelope, a localization that was suggested to depend on the nuclear transport factor importin β. In this thesis, by usage of rapamycin-induced dimerization assays, a localization to the INM could be confirmed. However, INM targeting of LRRC59 is independent of importin β. Additionally, the extraluminal region of LRRC59 does not contain a nuclear localization signal. Instead, the efficiency of INM targeting of LRRC59 was shown to depend on the size of the extraluminal domain, suggesting passive diffusion of LRRC59 to the INM by peripheral channels of the NPC. In addition to LRRC59, the ER located integral membrane proteins Sec61β, DDOST and Ube2j1 were shown to reach the INM mainly by passive diffusion. The INM targeting of these proteins were not only dependent on the size of the extraluminal domain but also on the geometry of the inserted tags in the rapamycin-induced dimerization assay. These results suggest that some ER-membrane proteins might reach the INM by diffusion as long as they fulfill the size restriction to pass the peripheral channels of the NPC.2020-06-2