Nuclear Pore Complex in Yeast - the Role of FG-repeats in Structure and Transport.

Genes are contained and organised in the nucleus which is separated from the rest of the cell by the membranous nuclear envelope. The nucleus communicates with the rest of the cell through channels in the nuclear envelope called nuclear pore complexes (NPCs). The NPCs have a pivotal role in controlling nuclear functions such as expression of genes and replication of DNA. They are massive, highly complex protein structures. Molecules that travel through them have to be carried by transport proteins. We have a good knowledge of the different transport carriers and how they interact with their cargoes and their control proteins. However, despite identifying most of the proteins that make up the NPC and understanding its architecture to a certain degree we do not know how transport carriers and their cargoes are propelled through the NPC channel. One reason for this is that NPC structure has mostly been determined using amphibian oocyte nuclear envelopes which are suited for electron microscopy (EM) studies but are difficult to manipulate experimentally. However model organisms such as yeast where it is easy to mutate NPC proteins and work out there role in transport have not been accessible to structural studies. Therefore we have developed a number of advanced high resolution imaging methods aimed at determining the structure of yeast NPCs. We have collaborated with a group in the USA which has a comprehensive collection of yeast cells where different combinations of NPC protein genes have been mutated. These mutations have specific effects on transport of different types of cargoes. We have a unique facility for determining the 3D structure of large protein complexes using various EM methods. This includes high resolution scanning EM for looking at the surface of the NPC at nanoscale resolution where we can detect individual proteins. We can link antibodies to small gold markers and use these to locate specific proteins in the structure. We will use transmission EM to determine the 3D structure of the NPC by 'EM tomography' and can use antibody-gold labelling to locate NPC proteins. We will use these methods to determine what effect mutations of the NPC proteins have on NPC structure. In particular we will look at a group of proteins that are known to be essential for and directly involved in transport. Our collaborators have also constructed genes expressing cargo molecules that are tagged with a fluorescent protein called GFP. The GFP tag allows the cargo molecules to be followed in live cells by fluorescence light microscopy but also provides a convenient tag for identifying the protein in the EM by antibody-gold labelling. Therefore we can follow the progress of cargo molecules through the NPC. Our plan is to follow the route of transport of a particular cargo through the NPC and see how the mutations affect this route. Such experiments will tell us what effect removing particular parts of specific proteins has on the structure of the NPC and will tell us how these proteins contribute to NPC structure. We will look at essential proteins involved in the transport of different cargoes. We will then see how the interaction of these specific cargoes with the NPC is altered and how their transport is affected. In the past 10 years our understanding of nuclear transport has made phenomenal progress and we understand how transport carriers interact with cargoes and control proteins exquisitely. Moreover a network of related pathways involved in transporting different types of cargoes have been discovered and characterised. All these pathways converge on the NPC. The NPC however remains a bit of a 'black box'. We know what it is composed of, but we don't know how the components fit together or how they interact with the transport carriers during transport. The work proposed here addresses this and could provide a breakthrough in understanding how this pivotal part of a key cellular process occurs.

Nuclear pore complexes (NPCs) control the transport of macromolecules to and from the nucleus. We have deep understanding of the soluble receptors that carry cargoes through the NPCs, as well as the proteins that control their interactions. However, we do not understand how the NPC structure, upon which all transport pathways converge, is functionally involved in the transport process. One reason for this is that our understanding of NPC structure has come primarily from electron microscopy (EM) of amphibian oocyte nuclear envelopes which is not an experimentally tractable system. Conversely, model organisms such as budding yeast where mutations to NPC proteins can be readily introduced have not been extensively studied by EM. We have developed methods using high resolution field emission scanning EM (feiSEM) and EM tomography to study yeast NPC structure. We have access to a comprehensive collection of yeast nucleoporin mutants enabling us to combine structural and molecular studies. In particular we will initially focus on a family of nucleoporins which contain phenylalanine-glycine (FG) repeats which are known to be directly involved in translocation. It has been shown that deletion of specific combinations of FG domains has effects on specific transport pathways. Our aim is to determine what effects these mutations have on NPC structure then correlate these to functional effects. We will also express tagged protein cargoes in the wild type and mutant backgrounds and determine how the deletions perturb the route of transport through the NPC. We will use a range of advanced EM techniques and sample preparations methods to give us a comprehensive picture of yeast NPC structure, with or without mutations and/or cargoes. We have developed cryo methods both for fixation and visualisation of samples by both feiSEM and TEM and EM tomography as well as more conventional methods so we can be confident of our interpretation of the structural information.