The Structure and Function of GLFGs in the Nuclear Pore Complex of Yeast

Abstract

The nuclear pore complex (NPC) is a large multiprotein complex which perforates the nuclear envelope. The NPC is made up of nuclear pore proteins (Nups), one third of which are phenylalanine-glycine (FG) containing. The NPC has a role in controlling movement of molecules between the nucleus and the cytoplasm. The FG Nups fill the NPC’s centre and regulate translocation. There are many different proposed models of how FG Nups may regulate translocation from them reeling cargo complexes into the NPC to inter FG repeats binding to create a gel-like meshwork into which specific cargo can enter and translocate. Using transmission electron microscopy the glycine-leucine-phenylalanine-glycine (GLFG) domains of Nups are mapped, showing a cytoplasmic bias within the wild type (WT) NPC and also in FG domain deletion mutants. FG deletion mutants have higher percentages of GLFG labelling towards the NPC edge than WT and lower percentages towards the middle than WT. GLFG domain labelling is also observed ‘reaching’ to membrane structures from the NPC. Serial sectioning of individual NPCs confirmed that individual NPCs had different distributions of GLFG labelling, which was on the nucleoplasmic or cytoplasmic side, or on both sides. Mutants which are defective in the nucleotide exchange activity of the RanGEF, Prp20, have a deficiency of the active RanGTP molecular switch. This causes a shift in the GLFG labelling from the cytoplasmic side towards the nucleoplasmic side. Similarly the import of Kap121-dependant import cargo causes a shift from cytoplasmic to nucleoplasmic labelling. This is observed as the cargo reaches the midplane of the NPC. Field emission scanning electron microscopy shows GLFG labelling to be associated with filaments (cytoplasmic, internal and nucleoplasmic) and possibly also the transporter. Finally, a model based on the shift in GLFG labelling is developed. This model suggests that there is a collapse and ‘reel in’ of import cargo as in the reversible collapse model, there is then a restructure of GLFG domains into the nucleoplasm due to potentially passing cargo on.