Figure 2

Nuclear import of ASV-BSA and SV40-BSA substrates; import of ASV-BSA does not require the Impα-Impβ pathway. A. Digitonin-permeabilized HeLa cells were incubated in the presence of complete transport mixture containing the ASV-BSA conjugate, the SV40-BSA conjugate, or Texas red-labeled BSA (TR-BSA). Top panels: Visualization of Texas red conjugates by fluorescence microscopy. Bottom panels: Differential interference contrast (DIC) microscopy of the same field to show preservation of cell integrity. B. Digitonin permeabilized HeLa cells were untreated (no addition), treated with 50 μg/ml wheat germ agglutinin (WGA), or 50 units/ml apyrase (Apyrase) prior to incubation with complete transport mixture containing either the ASV-BSA or the SV40-BSA import substrates. C. Free NLS peptides were added to the import reactions in molar excess of the import substrates as indicated. "Self" signifies competition with the homologous peptides; "Cross" indicates competition for ASV-BSA import by excess SV40TAg NLS peptide or competition for SV40-BSA import by excess ASV NLS peptide. The left column panels show import in the absence of competitor peptides. D. Depletion of ASV-BSA import factor(s) from cytosolic extracts. All assays included Texas-Red labeled ASV-BSA except that shown in the lower left hand corner (panel 4) which included Texas-Red labeled SV40-BSA. Cytosol was either not treated (1; no depletion) or pretreated with glutathione-beads that bound GST alone (2) or fusion proteins of GST plus IN(1–207) which lacks the IN NLS (3), full-length IN(1–286) (5), or a fragment of IN(201–236) that contains the IN NLS (panels 4 and 6).