On the mechanism of transport of Inner Nuclear Membrane Proteins

The nucleus is usually the biggest, round-shaped organelle in the cell, which contains numerous proteins and nucleic acids and protects the DNA. Nuclear components are contained within the boarders of Nuclear Envelope (NE), a double membrane system, formed by the fusion of Outer Nuclear Membrane (OMN) and Inner Nuclear Membrane (INM). The ONM is continuous with the Endoplasmic Reticulum (ER) and shares its molecular composition, while the INM composition is distinct. In the place, where INM and ONM meet, pores are formed and huge protein assemblies are inserted – the Nuclear Pore Complexes (NPCs). This special position of NPCs in the cell gives them a unique function – they create a barrier for molecules that should accumulate in the nucleus, while allowing other molecules to pass. In this thesis I have investigated what is the mechanism that keeps the INM and ONM protein composition distinct. Our hypothesis was that the difference between INM and ONM composition relates to the question: which proteins are permitted to cross the NPC and which are not. To study this phenomenon we have chosen an INM protein from yeast Saccharomyces cerevisiae, Heh2p, and created a set of Heh2p based reporters fused to GFP for detecting their localization using confocal fluorescence microscopy and super-resolution microscopy (STORM). We conclude that Heh2p needs an NLS and a linker domain for efficient transport to the INM, and we provide evidence that the transport of membrane proteins takes place with the hydrophobic domains embedded in the membrane environment. Using an assay, in which the reporters were trapped at distinct positions of the NPC, we mapped the route of membrane proteins through the NPC