Isolation of the Yeast Nuclear Pore Complex

Nuclear pore complexes (NPCs) have been isolated from the yeast Saccharomyces. Negative stain electron microscopy of the isolated NPCs and subsequent image reconstruction revealed the octagonal symmetry and many of the ultrastructural features characteristic of vertebrate NPCs. The overall dimensions of the yeast NPC, both in its isolated form as well as in situ, are smaller than its vertebrate counterpart. However, the diameter of the central structures are similar. The isolated yeast NPC has a sedimentation coefficient of '-,310 S and an Mr of ',,66 MD. It retains all but one of the eight known NPC proteins. In addition it contains as many as 80 uncharacterized proteins that are candidate NPC proteins. N 'UCLeAR pore complexes (NPCs) 1 occur at circular apertures in the nuclear envelope where the inner and outer nuclear membranes are joined, and mediate nucleocytoplasmic exchanges. Their ultrastructure has been well studied, and mapped to a resolution of better than 10 nm in Xenopus. They are cylindrical supramolecular assemblies displaying octagonal symmetry, with their eightfold axes perpendicular to the plane of the nuclear envelope (for review see Akey, 1992). NPCs are found in all eukaryotic cells and their general morphology appears to be highly conserved between evolutionary divergent phyla. The role of NPCs in nucleocytoplasmic transport has been studied intensively in recent years. The presence of the NPC limits the functional size of passive diffusion across the nuclear envelope to ,~9-10 rim. However, proteins and RNAs larger than this can be rapidly transported through the center of each NPC by an active, vectorial, signal-dependent process (for reviews see Franke, 1974; Maul, 1977; Gerace and Burke, 1988; Miller et al., 1991; Forbes, 1992). The detailed ultrastructure of vertebrate NPCs has been well documented, particularly in isolated nuclear envelope and NPC-lamina fractions, which are free of material that normally obscures the NPC. Ultrastructural analysis of nuclear envelopes from Xenopus oocytes has shown that the NPC is composed of several distinct structural regions. The core of the NPC is built from three coaxial rings, each around 120 nm in diameter; the outer and inner rings are coplanar with the outer and inner nuclear membranes, respectively, and have an eightfold symmetry similar to that of the central ring, which is made of eight radial spokes. Each spoke is divided perpendicular to its axis into three doPlease address all correspondence to Dr. G. Blobel, Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021. 1. Abbreviations used in this paper: NPCs, nuclear pore complexes; SPBs, spindle pole bodies. mains. Residing centrally within and supported by this spoke-ring complex is the plug, believed to be a coaxial tube (termed the "transporter") through which macromolecules are actively translocated. Pairs of radial arms interconnect adjacent spokes within the lumen of the nuclear envelope, to form a continuous ring coplanar with and surrounding the central spoke ring (Unwin and Milligan, 1982; Akey, 1989, 1990; Hinshaw et al., 1992; Akey and Radermacher, 1993). Eight perpendicular filaments extend from both the nuclear and cytoplasmic surfaces. The nuclear filaments are linked by a distal ring to form the so-called "baskets" (Ris, 1990, 1991; Jarnik and Aebi, 1991; Goldberg and Allen, 1992). Little is known about the composition of the NPC. The biochemical characterization of NPC proteins has used material derived primarily from rat liver nuclei. Procedures producing partially enriched fractions of NPCs from these nuclei have been described (Dwyer and Blobel, 1976), though intact NPCs have not been separated from the attached lamina. Generally, proteins associated with the NPC have been identified in vertebrates either immunochemically or by subfractionation of nuclear envelopes (Gerace et al., 1982; Davis and Blobel, 1986, 1987; Snow et al., 1987; Park et al., 1987; DabauvaUe et al., 1988; Wozniak et al., 1989; Radu et al., 1993). Only four such mammalian proteins have been molecularly cloned and sequenced to date (Wozniak et al., 1989; Starr et al., 1990; Cordes et al., 1991; CarmoFonesca et al., 1991; Sukegawa and Blobel, 1993; Radu et al., 1993). A number of monoclonal antibodies that identify members of a family of NPC proteins in mammals also cross-react with homologous proteins in Xenopus and the yeast Saccharomyces (Davis and Blobel, 1986, 1987; Snow et al., 1987; Park et al., 1987; Dabauvalle et al., 1988; Featherstone et al., 1988; Aris and Blobel, 1989; Davis and Fink, 1990; Wente et al., 1992; Loeb et al., 1993). This cross-reactivity has been used to molecularly clone and sequence five yeast NPC proteins (Davis and Fink, 1990; Wente et al., 1992; Loeb et al., 1993), with a sixth having © The Rockefeller University Press, 0021-9525/93/11/771/13 $2.00 The Journal of Cell Biology, Volume 123, Number 4, November 1993 771-783 771 on July 8, 2017 jcb.rress.org D ow nladed fom been identified independently (Nehrbass et al., 1990). Two of these proteins were subsequently identified using genetic screens (Wimmer et al., 1992). Though little is known about the detailed structure of yeast NPCs, they have similar functions to those of higher eukaryotes (Forbes, 1992). Biochemical approaches of the type successfully used in the isolation of vertebrate NPC proteins have proven more difficult in yeast. However, the production of large quantities of yeast nuclei is relatively straightforward and some progress in their subfractionation has been reported (Rozijn and Tonino, 1964; Kilmarfin and Fogg, 1982; Hurt et al., 1988; Aris and Blobel, 1988, 1989; Allen and Douglas, 1989; Cardenas et al., 1990; Rout and Kilmartin, 1990). Here we describe a large scale, high yield biochemical procedure producing a highly enriched NPC fraction, that has revealed over 80 distinct candidate NPC proteins. Materials and Methods Buffers and Solutions NPC buffer: 10 mM bisTris-C1, pH 6.50, 0.1 mM MgC12, 1.0% sodium taurodeoxycholate (Sigma Chem. Co., St. Louis, MO), 10 #g/ml RNase A (Sigma Chem. Co.), 0.5 mM DTT. bt buffer: 10 mM bisTris-C1, pH 6.50, 0.1 mM MgC12. bt-DMSO buffer: 10 mM bisTris-Cl, pH 6.50, 0.1 mM MgC12, 20% DMSO. Solution P: 87.0 mg PMSF plus 1.5 nag pepstatin A, dissolved in 5 ml dry absolute ethanol. Preparation of a Highly Enriched Nuclear Pore Complex Fraction All solutions contained a 1:1,000 dilution of solution P unless otherwise stated. The yeast strain Saccharomyces uvarum (NCYC 74, ATCC 9080), considered a strain of S. cerevisiae (Mortimer and Johnston, 1986), was used due to the ease with which it can be spheroplasted (Eddy and Wdliamson, 1957). Crude and enriched nuclear fractions were prepared by using the method described in Rout and Kilmartin (1990, 1993) (modified from Rozijn and Tonino, 1964), except that Triton X-100 was omitted from the spheroplast lysis solution. Nuclear lysis and the separation of the lysate over a stepped sucrose gradient were exactly as described, with the crude NPC fraction being recovered from the S/1.75 M interface (Rout and Kilmartin, 1990, 1993). The enriched NPC fraction was produced as follows. The protein concentration of the S/1.75 crude NPC fraction (above) was determined by using the Bradford protein assay (see below). Five volumes of this fraction were then diluted by the addition of one volume of ice cold NPC buffer, a 1:200 dilution of solution P, and a final concentration of 0.045 rag of heparin (sodium salt; Sigma Chem. Co.) per 1.0 mg of S/1.75 M fraction protein. The mixture was vortexed thoroughly, incubated for 15 rain at 10*C, and centrifuged to remove the froth (700 g, 4 rain, 4°C) before being loaded onto precooled SW55 tubes (Beckman Instrs., Fullerton, CA) (1.2 ml per tube), each containing 0.5 ml of 2.50 M sucrose-bt (refractive index 1.4533), 1.5 ml of 1.85 M sucrose-bt + 0.2% Triton X-100 (refractive index 1.4225), and 1.5 ml of 1.45 M sucrose-bt + 0.2% Triton X-100 (refractive index 1.4032). It should be noted that making all the sucrose solutions discussed in this method by their refractive index (measured at room temperature) was important for the success of the procedure. The sample layer was then overlayered with 0.7 ml of bt-DMSO and the tubes were centrifuged at 237,000 g~, for 5 h at 4°C. The tubes were unloaded from the top. The first 1.5 ml was termed the S fraction and the second 1.5 ml the S/1.45 fraction, as they contained the supernatant and the superuatant/l.45 M interface, respectively. The enriched NPC fraction was recovered in 1.5 mi from the 1.45 M/ 1.85 M interface, called the 1.45/1.85 fraction. The final 0.8 ml contained the 1.85 M/2.50 M interface (installed for diagnostic purposes) and was termed the 1.85/2.5 fraction. To produce the highly enriched NPC fraction, the pooled 1.45/1.85 enriched NPC fraction was diluted with an equal volume of bt-DMSO buffer and a 1:500 dilution of solution P. 4-mi aliquots of this were each overlayered onto a Beckman SW28 centrifuge tube containing a 5-ml cushion of"1.75 M" sucrose bt-DMSO + 0.01% Tween-20 (refractive index 1.4495) and 29 mi of a continuous linear gradient of "1.20 M" sucrose (refractive index 1.4220) to "1.00 M" sucrose (refractive index 1.4120) in bt-DMSO + 0.01% Tween-20. All the tubes were then centrifuged in a Beckman SW28 rotor at 104,000 gay for 24 h at 4°C. After centrifugation a faint white band was visible around the "1.20 M"/"1.75 M" interface in each tube; its position was marked. Each gradient was unloaded from the top; the first 9 ml was termed fraction #1 and the second 9 ml, fraction #2. The third 9 mi, fraction #3, was collected to within •5 mm above the marked band. The fourth fraction was the 10 ml containing the marked band and the "1.20 M'/"1.75 M" interface. It was this fraction #4 that contained the NPCs and was referred to as the highly enriched NPC fraction. Fraction #5 was the final 1 ml collected after vortexing to resuspend any pellet. This final gradient, though reproducible, also had the greatest tendency for variability and so great care was needed during its preparation and running. For ion exchange chromatography and HPLC analysis, the proteins in fraction #4 or in the enriched nuclei were precipitated for I h with methanol (90% final concentration) at -20°C and resuspended in 10 mM Na-MES, pH 6.5, 100 mM DTT, 1.0% SDS. They were then diluted with 9 vol of 20 mM Na-MES, pH 6.5, 7 M urea, 1.0% Triton X-100 and batch incubated for 1 h at room temperature with S-sepbarose resin (Pharmacia, Uppsala, Sweden) preequilibrated with column buffer (20 mM Na-MES, pH 6.5, 7 M urea, 1.0% Triton X-100, 0.1% SDS, 1 mM DTT). Unbound proteins were washed out of the resin with column buffer. Bound proteins were eluted with 1 M NaC1 in column buffer. The two fractions were further separated by reverse phase HPLC, essentially as described (Pain et al., 1990; Wozniak, R., personal communication). Gel Electrophoresis and Immunoblotting Protein samples for SDS-PAGE (Laemmli, 1970) were either diluted directly with 0.5 vol of 3 × sample buffer, or ware concentrated by ultracentrifugation or by precipitation for 1 h with methanol (90% final concentration) at -20"C or TCA at 0*C, before resuspension in sample buffer. The samples were then incubated at 90"C for 10 rain. All the gels presented here contained a 5-16% acrylamide gradient, and high and low molecular mass standards were used (BioRad Labs., Hercules, CA). Proteins were visualized after electrophoresis by staining with Coomassie brilliant blue, by silver staining, or by a combination of the two. Immunoblots were prepared essentially as described in Towbin et al. (1979). They were then stained using the appropriate primary antibody as described in Wente et al. (1992). The only exception was with the monoclehal antibody MAb306, in which the Tween-20 was omitted from the blot buffer for all the incubations and washes. The blots were either exposed at -70°C on prettashed medical X-ray film or, in the case of quantitative immunoblotting for estimations of isolated NPC yields, the immtmoblots obtained with MAb350 and MAb192 were exposed on a phosphor screen and the signals from NUP63 and NUP49 quantified using a PhosphorImager with lmageQuant software on an IBM PC (Molecular Dynamics, Inc., Sunnyvale, CA). To reduce variations and nonlinearities due to various immunoblotting artifacts (for review see Harlow and Lane, 1988), the signal intensifies were then combined to obtain an overall yield. Electron Microscopy and Image Processing For negative stain electron microscopy, 15 #1 aliquots of the sample were diluted with 15 #1 of bt-DMSO buffer and centrifugad onto the surface of a glow discharged carbon-formvar 300 mesh copper grid at 1,800 g for I h at 4°C. The grid was then washed with bt-DMSO, and fixed with 3.7% formaldehyde, 0.5 % glutaraldehyde in bt-DMSO for 20 rain at 250C. After washing with bt-DMSO, the grids ware negatively stained with either 4 % uranyl acetate or 2 % potassium phosphotungstate (pH 7). Magnifications were calibrated using uniform latex microspheres of defined diameter (Duke Scientific Corp., Pale Alto, CA). Pairs of micrographs taken at 0* and 60 * ware made with a manually adjusted goniometer stage. Estimates of the thickness of NPCs (x) were made from measuring the NPC diameter parallel to the direction of tilt at 0* (D1) and at 60 ° (D2) from the formula: 1.4{/)1 [D2 cos 60*]} x = sin 60* The adjustment factor of 1.4 is an approximate compensation for z-axis compression in negative stain (Hinsbaw et al., 1992). For the image analysis, seven micrographs of fields of uranyl acetate negatively stained isolated yeast NPCs were scanned with a modified JoyceThe Journal of Cell Biology, Volume 123, 1993 772 on July 8, 2017 jcb.rress.org D ow nladed fom Loebl flatbed densitometer. The resulting image arrays were compressed a factor of three for display and particle selection. In all, 75 NPCs were chosen for further analysis based on the preservation of particle circularity and the appearance of subunit morphology. As the particle symmetry was not known a priori, the analyses were conducted as follows. Each particle was used as a reference in two complete alignment passes which encompassed rotational, translational, and flip/flop analyses using SPIDER (Frank et al., 1981; Akey, 1989). An average was then computed with and without enforced eightfold symmetry for each reference particle. Overall, eight particles were discarded as not aligning consistently. Therefore 67 particles were used for each average. Samples for thin section electron microscopy were fixed in t.25% glutaraldehyde, 0.2% tannic acid for 30 min at 25°C in their original buffer (usually sucrose-bt), and then for 16 h at 4°C in the same fixative buffered with 0.05 M potassium phosphate pH 7.0. After postfixation in 1% osmium tetroxide the samples were dehydrated with graded ethanol and embedded in Epon. Sections were stained with uranyl acetate and lead citrate. Magnifications were controlled for by measurements from structures of known dimensions (microtubules and SPBs). All samples were viewed with a JEOL100CX electron microscope at 80 kV and photographed with Kodak electron microscope film. Estimation of the Sedimentation Coeffwient, Diffusion Coefficient, and Molecular Mass of the NPC Enriched and highly enriched NPC fractions suitable for the following measurements was made as normal, except that the Triton X-100 and the Tween-20 added to the gradients (above) was replaced with octyl glucoside (Calbioehem Corp., La Jolla, CA) at the same concentrations. Under those conditions octyl glucoside would not form micelles, which might otherwise have interfered with the various measurements below. Yeast ribosomes prepared from yeast spheroplast homogenate (gift of C. Strambio de Castillia) were measured in parallel with the NPCs. The sedimentation coefficients of both the NPCs and ribosomes were determined essentially as described by Griffith (1986). The enriched NPC fraction was dialyzed overnight at 4°C into bt-DMSO, 0.1% Triton X-100, 1:1,000 solution P, and both this and the ribosome standard were separately overlayered onto Beckman SW41 centrifuge tubes containing 11 mi linear gradients of 10--40% sucrose, 10 mM bisTris-C1, pH (~50, 0.3 mM MgC12, 0.5 mM K-acetate, 0.01% octyl glucoside, 1:1,000 solution P, and then centrifuged at 202,000 g~ and 5°C for varying times. The gradients were unloaded manually from the top and collected as 15 equal fractions of 733 /~1. The linearity of all the gradients (an important assumption for the validity of the calculations) was confirmed after centrifugation by refractometric measurement of the sucrose concentrations of the collected fractions. The protein composition of the fractions was analyzed by SDS-PAGE and laser densitometry to determine quantitatively the peak fraction in each case. Negative stain EM was also performed on the fractions from one of the NPC gradients. Knowing the time and speed of the run and the sucrose concentration of the peak fraction, the time integral tables of both McEwen (1967) and Griffith (1986) could be used to calculate s-values. The diffusion coefficients of the NPCs and ribosomes were measured using a Biotage dp801 molecular size detector, with software supplied by the manufacturers run on a Texas Instruments Travelmate 2000 PC. Both the NPC sample and ribosome standards were usually diluted 1:50 in 10 mM bisTris-Cl, pH 6.50, 0.1 mM MgC12, 0.35 mM KCI. The density of NPCs was estimated by density sedimentation of the highly enriched NPC fraction and the ribosomes over linear 0.6 mi gradients of 1.75-2.50 M sucrose-bt, 0.01% octyl glucoside, 1:1,000 solution P at 237,000 g~ for 27 h at 20°C in a Beckman SW55Ti rotor. The resultant fractions were analyzed by refractometry and SDS-PAGE. The molecular masses (M) were calculated from the sedimentation (s) and diffusion (D) coefficients using the formula: