It has recently been shown that large karyophilic proteins are transported across the nuclear envelope in amphibian oocytes. In consideration of this, the present experiments were performed to (a) identify the specific sites within the envelope through which transport occurs and (b) determine if molecular size is a limiting factor in the transport process. The following experimental procedure was employed: Colloidal gold particles, varying in size from ~20 to 170 A in diameter were coated with nucleoplasmin, a 165,000-mol-wt karyophilic protein, which is known to be transported through the envelope. The coated gold particles were microinjected into the cytoplasm of Xenopus oocytes, and the cells were fixed 15 min and 1 h later. The intracellular localization of the gold was then determined with the electron microscope. It was found that nucleoplasmin-coated particles readily enter the nucleus. On the basis of the distribution of the particles associated with the envelope, we concluded that transport occurs through the nuclear pores. Furthermore, the size distributions of the gold particles present in the nucleus and cytoplasm were not significantly different, indicating that the envelope does not discriminate among particles with diameters ranging from 50 to 200 ,g, (the dimensions including the nucleoplasmin coat). Colloidal gold coated with trypsin-digested nucleoplasmin (which lacks the polypeptide domain required for transport) or exogenous polyvinylpyrrolidone were largely excluded from the nucleus and showed no evidence of transport.Exogenous macromolecules appear to enter the nucleus by diffusing through central channels located within the nuclear pores (6). In amphibian oocytes it has been estimated that the channels available for passive diffusion are ~90 A in diameter (20). The rate of diffusion through these regions is inversely related to the size of the permeating substance. For example, in oocytes, exogenous molecules with hydrodynamic radii of ~ 15 A rapidly diffuse into the nucleus, whereas substances with radii of -45 ~, diffuse at extremely slow rates, if at all (20).Extrapolating to endogenous molecules, it is reasonable to assume that small proteins enter the nucleus by passive diffusion through the pores (although some form of facilitated uptake cannot be excluded). On the other hand, large nuclear proteins, which have a limited capacity for diffusion through 90 ~, pores, are presumably transported across the envelope. Evidence for transport has been obtained in two recent studies. Feldherr et al. (9) demonstrated that RN1, a 150,000-mol-wt nuclear protein found in Rana pipiens oocytes, enters the nucleus approximately 20 times faster than can be accounted for by diffusion through the pores. Dingwall et al. (3) showed that nucleoplasmin, a 165,000-mol-wt pentamer, is transported across the envelope. The transport of nucleoplasmin is prevented if a 12,000-mol-wt tail region, present on each of the monomeric subunits, is removed by protease digestion.This report is concerned mainly w...

Nuclear transport factor 2 (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1p and vertebrate p62. NTF2 is required for efficient nuclear protein import and has been shown to mediate the nuclear import of RanGDP. We have used the crystal structures of rat NTF2 and its complex with RanGDP to design a mutant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is reduced while wild-type binding to RanGDP is retained. The 2.5 A resolution crystal structure of W7A-NTF2 is virtually superimposable upon the wild-type protein structure, indicating that the mutation had not introduced a more general conformational change. Therefore, our data suggest that the exposed side-chain of residue 7 is crucial to the interaction between NTF2 and xFxFG repeat-containing nucleoporins. Consistent with its reduced affinity for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less strongly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated with W7A-NTF2 binds less strongly to the central channel of nuclear pore complexes than wild-type NTF2-coated gold. Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing direct evidence that an interaction between NTF2 and xFxFG repeat-containing nucleoporins is required to mediate the nuclear import of RanGDP.

Abstract. To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors.It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals.