Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex.
Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing. Therefore, we have developed methods to accurately determine cargo positions within the NPC. Cargos were instantly trapped in transit by high-pressure freezing, optimally preserved by low-temperature fixation and then localized by immunoelectron microscopy. A statistical modelling approach was used to identify cargo distribution. We found import cargos localized surprisingly close to the edge of the channel, whereas mRNA export factors were at the very centre of the NPC. On the other hand, diffusion of GFP was randomly distributed. Thus, we suggest that spatially distinguished pathways exist within the NPC. Deletion of specific FG domains of particular NPC proteins resulted in collapse of the peripheral localization and transport defects specific to a certain karyopherin pathway. This further confirms that constraints on the route of travel are biochemical rather than structural and that the peripheral route of travel is essential for facilitated import.