Fig. 4. (A) The rate of facilitated translocation is determined not only by properties of the receptor, but also by those of the cargo. Three different inert proteins, namely GFP, MBP and an MBP dimer (2×MBP), were each fused to an IBB domain (a potent Impβ-dependent import signal). The GFP fusion was detected through its intrinsic fluorescence. Fluorescent MBP fusion proteins had been labelled with Alexa 488 maleimide. The fluorescent fusion proteins (0.5 µM final) were pre-bound to stoichiometric amounts of Impβ and their import into nuclei of permeabilized cells was allowed in the presence of Ran and an energy-regenerating system. The distribution of the fusions was determined by confocal fluorescence microscopy. Panels show the reactions after 9 s of import. Even though the three substrates were linked to identical import signals and imported under identical conditions by the same receptor, their import rates were strikingly different. Thus, the translocation rate is not only determined by the receptor and the type of signal, but also by the ‘rest’ of the cargo domain. (B) Enlarged field of the IBB–2×MBP sample from (A). Clear NPC staining is evident even against the high background of cytoplasmic substrate.