Comparative Spatial Localization of Protein-A-Tagged and Authentic Yeast Nuclear Pore Complex Proteins by Immunogold Electron Microscopy Birthe Fahrenkrog,* ,1 John P. Aris,† Eduard C. Hurt,‡ Nelly Pante ´,¶ ,2 and Ueli Aebi* ,3 *M. E. Mu ¨ ller Institute for Structural Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland; †Department of Anatomy and Cell Biology, Health Science Center, University of Florida, Gainesville, Florida 32610; ‡Biochemie-Zentrum Heidelberg, University of Heidelberg, D-69120 Heidelberg, Germany; and ¶Institute of Biochemistry, Federal Institute of Technology, CH-8092 Zu ¨ rich, Switzerland Received November 23, 1999, and in revised form January 21, 2000 The nuclear pore complex (NPC) mediates protein and RNP import in and RNA and RNP export out of the nucleus of eukaryotic cells. Due to its genetic tractabil- ity, yeast offers a versatile system for investigating the chemical composition and molecular architecture of the NPC. In this context, protein A tagging is a com- monly used tool for characterizing and localizing yeast NPC proteins (nucleoporins). By preembedding anti- protein A immunogold electron microscopy (immuno- gold EM), we have localized two yeast nucleoporins, Nsp1p and Nic96p, in mutant yeast strains recombi- nantly expressing these nucleoporins tagged with four (Nsp1p) or two (Nic96p) IgG binding domains of pro- tein A (i.e., ProtA-Nsp1p and ProtA-Nic96p). We have compared the location of the recombinant fusion pro- teins ProtA-Nsp1p and ProtA-Nic96p (i.e., as specified by their protein A tag) to the location of authentic Nsp1p and Nic96p (i.e., as defined by the epitopes recognized by corresponding nucleoporin antibodies) and found all of them to reside at the same three NPC sites. Hence, recombinant expression and protein A tagging of the nucleoporins Nsp1p and Nic96p have not caused any significant mislocation of the fusion pro- teins and thus enabled mapping of these two yeast nucleoporins at the ultrastructural level in a faithful manner.  2000 Academic Press Key Words: Nic96p; Nsp1p; nuclear pore complex; nucleoporin; protein A; yeast. INTRODUCTION Anchored in the double membrane of the nuclear envelope (NE), the vertebrate nuclear pore complex (NPC) is a supramolecular assembly of proteins— and possibly RNAs(?)—with an estimated molecular mass of 125 MDa that mediates the bidirectional molecular trafficking between the cytoplasm and the cell nucleus (for reviews see Izaurralde and Adam, 1998; Mattaj and Engelmeier, 1998; Ohno et al., 1998; Adam, 1999). A consensus model of the three- dimensional (3-D) architecture of the NPC has evolved from extensive electron microscopic studies on amphibian oocytes (for reviews see Pante ´ and Aebi, 1996a; Stoffler et al., 1999). Accordingly, the NPC is built of an octagonal central framework (also called the spoke complex), sandwiched between a cytoplasmic and a nuclear ring moiety. The cytoplas- mic ring is decorated by eight cytoplasmic fibrils, whereas the nuclear ring is capped by a highly dynamic basket-like assembly. The central frame- work embraces the central channel, which is in- volved in mediating signal-dependent nucleocytoplas- mic transport (for reviews see Pante ´ and Aebi, 1996a; Stoffler et al., 1999). The central channel appears often plugged by a ‘‘particle’’ (called the ‘‘central plug’’ or ‘‘transporter’’) of yet poorly defined structure and function. Recent EM studies have revealed that yeast NPCs have a 3-D architecture very similar to that of vertebrate NPCs, although the yeast NPCs are about 15% smaller in their linear dimensions, so that their mass amounts to only about 60 MDa (Fahrenkrog et al., 1998; Yang et al., 1998). Moreover, judged from 3-D reconstructions of thin ice-embedded isolated yeast NPCs these appear to lack distinct cytoplasmic and nuclear ring moi- eties (Yang et al., 1998). The NPC is composed of 100 different proteins, called nucleoporins (Nups), in vertebrates and of 50 in yeast (reviewed in Pante ´ and Aebi, 1996a; Stoffler et al., 1999). Combining biochemical and genetic approaches and completion of the yeast genome project have yielded the identification and 1 Present address: EMBL, Meyerhofstr.1, D-69117 Heidelberg, Germany. 2 Present address: Department of Zoology, University of British Columbia, Vancouver B.C. V6t 1Z4, Canada. 3 To whom correspondence and reprint requests should be addressed. Fax: 0041/61/267-2109. E-mail: ueli.aebi@unibas.ch. Journal of Structural Biology 129, 295–305 (2000) doi:10.1006/jsbi.2000.4223, available online at http://www.idealibrary.com on 295 1047-8477/00 $35.00 Copyright  2000 by Academic Press All rights of reproduction in any form reserved.