Fig. 5. Imp4 is a functional nuclear import receptor and a cytoplasmic chaperone for the ribosomal protein S3a. (A) Nuclear import of Alexa 488-labelled rpS3a (0.5 µM) was performed with Ran, an energy-regenerating system and the indicated transport receptors (1.5 µM each). Imp4a, Imp5 and the Impβ/7 heterodimer were efficient in rpS3a import. Under these conditions, all three receptor species suppressed aggregation of rpS3a with the cytoplasmic remnants of the permeabilized cells. (B) Imp4a is necessary and sufficient to suppress precipitation of rpS3a in a cytoplasmic environment. The anti-precipitation assays contained 0.5 µM rpS3a in either buffer or a cytoplasmic HeLa extract depleted of importins. Note that rpS3a readily precipitates in a cytoplasmic environment when importins are absent. This aggregation was completely suppressed by re-addition of Imp4a (1.5 µM), while Imp5 (1.5 µM), and the Impα/β (1.5 µM) and Impβ/7 (0.75 µM) heterodimers show only a weak anti-precipitation activity towards this substrate. (C) Nuclear import of rpS3a was performed exactly as in (A), but importin-free cytosol (see Materials and methods) was included in the reaction. Under these conditions, only Imp4a was efficient as a chaperone and import mediator.