We have previously demonstrated [Rihs, H.‐P. and Peters, R. (1989) EMBO J., 8, 1479–1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T‐antigen are fused to the amino terminus of Escherichia coli beta‐galactosidase is dependent on both the nuclear localization sequence (NLS, T‐antigen residues 126–132) and a phosphorylation‐site‐containing sequence (T‐antigen residues 111–125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation‐site‐containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK‐II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK‐II sites at a distance of approximately 10–30 amino acid residues from the NLS. CK‐II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK‐II may exert this role by controlling the rate of nuclear protein transport.

Nuclear pore complexes (NPCs) act as effective and robust gateways between the nucleus and the cytoplasm, selecting for the passage of particular macromolecules across the nuclear envelope. NPCs are comprised of an elaborate scaffold that defines a ∼30nm diameter passageway connecting the nucleus and the cytoplasm. This scaffold anchors proteins termed ‘phenylalanine-glycine’ (FG)-nucleoporins, the natively disordered domains of which line the passageway and extend into its lumen1. Passive diffusion through this lined passageway is hindered in a size-dependent manner. However, transport factors and their cargo-bound complexes overcome this restriction by transient binding to the FG-nucleoporins2–10. To test whether a simple passageway and a lining of transport-factor-binding FG-nucleoporins are sufficient for selective transport, we designed a functionalized membrane that incorporates just these two elements. Here we demonstrate that this membrane functions as a nanoselective filter, efficiently passing transport factors and transport-factor–cargo complexes that specifically bind FG-nucleoporins, while significantly inhibiting the passage of proteins that do not. This inhibition is greatly enhanced when transport factor is present. Determinants of selectivity include the passageway diameter, the length of the nanopore region coated with FG-nucleoporins, the binding strength to FG-nucleoporins, and the antagonistic effect of transport factors on the passage of proteins that do not specifically bind FG-nucleoporins. We show that this artificial system faithfully reproduces key features of trafficking through the NPC, including transport-factor-mediated cargo import.

Lateral diffusion of bacteriorhodopsin and a lipid analogue has been measured in dimyristoylphosphatidylcholine bilayers as a function of temperature, phospholipid/protein (mol/mol; L/P) ratio, and aqueous phase viscosity. The protein lateral diffusion coefficients measured above the temperature at which the lipid gel-liquid/crystalline phase transition occurs (Tc) are combined with previously determined rotational diffusion coefficients to provide a test of the Saffman-Delbrfick equations [Saffman, P. G. & Delbrfick, M. (1975) Procm NatL Acade Sci USA 72,[3111][3112][3113]. Insertion of the diffusion coefficients into these equations enables the protein diameter to be calculated. The value of 4.3 ± 0.5 nm so obtained is in reasonable agreement with the known structure of bacteriorhodopsin. A 12-fold increase in the viscosity of the aqueous phase reduces protein lateral diffusion coefficients by 50%, which is also consistent with the Saffman-Delbruick equations. Both protein and lipid lateral diffusion coefficients decrease with decreasing L/P ratio above the T,. It is argued that, at a high L/P ratio, this effect is probably due to changes in membrane viscosity while, at a low L/P ratio, "crowding" effects (steric restrictions) and protein aggregation become important. When comparing diffusion measurements made in different systems, it is important to take the effect of the L/P ratio into account. When this is done, other published measurements offreely diffusing membrane proteins are in good agreement with the present results and the predictions of the Saffman-Delbruick equations. Below the Ta, the presence of protein enhances diffusion rates. The overall effect is to smooth out the large change in diffusion coefficient that occurs at the T,.It is probable that diffusion plays a crucial role in a number of membrane functions [e.g., receptor-mediated processes (1), electron transfer (2), and photoreception (3)]. Much has been learned about this field by the use of optical techniques developed for measuring diffusion of membrane components (for review, see refs. 4-6). Fluorescence microphotolysis (7) is a versatile means for measuring translational and rotational diffusion in single cells, isolated membranes, artificial membranes, and solution (8-13). Transient dichroism of intrinsic chromophores or triplet probes has provided data on the rotational diffusion of a variety of membrane proteins (14). In the case of triplet probes, rotational diffusion has also been measured by using phosphorescence (15,16), delayed fluorescence (17), and fluorescence-depletion signals (18).A fruitful approach for analyzing parameters that potentially might restrict and regulate mobility in cellular membranes is the study of artificial bilayer membranes. Membranes made from a single lipid species (19, 20) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

In this first application of optical single transporter recording (OSTR), a recently established technique for optically monitoring the activity of single transporters in membrane patches (Tschödrich-Rotter and Peters. 1998. J. Microsc. 192:114-125), the passive permeability of the nuclear pore complex (NPC) was measured for a homologous series of hydrophilic probe molecules. Nuclei were isolated from Xenopus oocytes and firmly attached to filters containing small cylindrical pores. Transport through membrane patches spanning filter pores was measured by scanning microphotolysis. Thus the permeability coefficients of single NPCs were determined for fluorescently labeled dextrans of approximately 4, 10, and 20 kDa. Dextrans of >/=40 kDa could not permeate the NPC. The data were consistent with a model in which the NPC contains a single diffusion channel. By application of established theories for the restricted diffusion through small pores, the diffusion channel was approximated as a cylinder with a radius of 4.4-6.1 nm (mean 5. 35 nm). Because the transport rate constant of the single NPC was known, the equivalent length of the channel could be also determined and was found to be 40-50 nm (mean 44.5 nm). The symmetry of the NPC implies that a singular component such as the diffusion channel is located at the center of the NPC. Therefore a common transport pathway apparently mediates both passive and signal-dependent transport. To test this hypothesis, measurements of signal-dependent transport and of the mutual effects signal-dependent and passive transport may exert on each other are in progress.

Abstract. The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation . We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase R (CK-II) site Si"ai2 . In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T'24 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import . Tí24 phosphorylation, which could be functionally simulated by a T-to-13 124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import C LOSE correlations between the nuclear cytoplasmic distribution ofcertain transcription factors (Lenardo and