Structure–Function Mapping of a Heptameric Module in the Nuclear Pore Complex

Published February 13, 2012 JCB: Article Structure–function mapping of a heptameric module in the nuclear pore complex Javier Fernandez-Martinez,1 Jeremy Phillips,3,4,5 Matthew D. Sekedat,2 Ruben Diaz-Avalos,6 Javier Velazquez-Muriel,3,4,5 Josef D. Franke,1 Rosemary Williams,1 David L. Stokes,6 Brian T. Chait,2 Andrej Sali,3,4,5 and Michael P. Rout1 1Laboratory of Cellular and Structural Biology and 2Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065 3Department of Bioengineering and Therapeutic Sciences, 4Department of Pharmaceutical Chemistry, and 5California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94158 6The New York Structural Biology Center, New York, NY 10027 he nuclear pore complex (NPC) is a multiprotein as- onto the complex, allowing us to identify regions that sembly that serves as the sole mediator of nucleocyto- stabilize the NPC’s interaction with the nuclear envelope plasmic exchange in eukaryotic cells. In this paper, membrane and connect the complex to the rest of the T Downloaded from we use an integrative approach to determine the structure NPC. Our data allow us to suggest how the Nup84 com- of an essential component of the yeast NPC, the Y600-kD plex is assembled into the NPC and propose a scenario heptameric Nup84 complex, to a precision of Y1.5 nm. for the evolution of the Nup84 complex through a series The configuration of the subunit structures was determined of gene duplication and loss events. This work demon- by satisfaction of spatial restraints derived from a diverse strates that integrative approaches based on low-resolution set of negative-stain electron microscopy and protein data of sufficient quality can generate functionally infor- jcb.rupress.org domain–mapping data. Phenotypic data were mapped mative structures at intermediate resolution. Introduction on March 28, 2012 Cells are comprised of thousands of highly organized, com- The NPC structural core is conserved, highly modular, plex, and dynamic subcellular macromolecular assemblies. To and is formed from eight symmetric spokes that connect to study how cells function, we need methodologies to determine form !ve coaxial rings: a membrane ring, two adjacent inner the structures, dynamics, and interactions of these assemblies rings, and two outer rings facing, respectively, the cytoplasmic and thus reveal how they give rise to the emergent properties and nucleoplasmic periphery (Alber et al., 2007b). Proteins of life. One such dynamic macromolecular assembly is the termed FG (phenylalanine–glycine) nups !ll the central chan- nuclear pore complex (NPC), the gatekeeper within the nuclear nel of the NPC and establish the permeability barrier (Peters, THE JOURNAL OF CELL BIOLOGY envelope (NE) that mediates the exchange of speci!c macro- 2009; Strambio-De-Castillia et al., 2010). Analysis of the fold molecules between the nucleoplasm and cytoplasm. Every composition of the NPC led to our proposal of the protocoat- NPC is formed by Y30 different proteins called nucleoporins omer hypothesis (Devos et al., 2004, 2006), which suggests a (nups), each present in multiple copies and connected in bio- common ancestry for the NPC and membrane-coating com- chemically stable subcomplexes that act as building blocks for plexes; they are thought to have evolved by divergent evolution the NPC (D’Angelo and Hetzer, 2008; Strambio-De-Castillia from a protocoatomer membrane–bending complex present in et al., 2010). the last eukaryotic common ancestor (DeGrasse et al., 2009; Field et al., 2011). Data from both vertebrates and the yeast Saccharomyces cerevisiae (Rout et al., 2000; Belgareh et al., 2001; Krull et al., J. Fernandez-Martinez and J. Phillips contributed equally to this paper. 2004; Alber et al., 2007b) indicate that the outer ring of the NPC Correspondence to Michael P. Rout: [email protected]; or A. Sali: sali@salilab .org Abbreviations used in this paper: BA, benzyl alcohol; CV, coefficient of varia- © 2012 Fernandez-Martinez et al. This article is distributed under the terms of an Attribution– tion; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrom- Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication etry; NE, nuclear envelope; NPC, nuclear pore complex; nup, nucleoporin; PAL, date (see http://www.rupress.org/terms). After six months it is available under a Creative protease accessibility laddering; PrA, Protein A; RMSD, root mean square devia- Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as de- tion; VCC, vesicle-coating complexes; YEPD, yeast extract peptone dextrose. scribed at http://creativecommons.org/licenses/by-nc-sa/3.0/). Supplemental Material can be found at: http://jcb.rupress.org/content/suppl/2012/02/09/jcb.201109008.DC1.html The Rockefeller University Press $30.00 J. Cell Biol. Vol. 196 No. 4 419–434 www.jcb.org/cgi/doi/10.1083/jcb.201109008 JCB 419 Published February 13, 2012 is comprised of a conserved assembly, which in vertebrates cor- kinds of biochemical and structural data. We apply this approach responds to a nonameric complex called the Nup107–160 com- to the yeast Nup84 complex, de!ning the positions and relative plex (Belgareh et al., 2001; Vasu et al., 2001; Loïodice et al., orientations of the individual components. The resulting struc- 2004) and in yeast corresponds to the Nup84 complex, which is ture allows us to visualize structure–function relationships that formed from seven proteins named Nup133, Nup120, Nup145c, provide insights into the assembly and evolution of the Nup84 Nup85, Nup84, Seh1, and Sec13 (Siniossoglou et al., 1996; complex and the NPC as a whole. Lutzmann et al., 2002). Sec13 is shared with the Sec13/31 COPII vesicle-coating complex (VCC), and both Seh1 and Sec13 have Results recently been found in a coating-related complex termed the Seh1-associated complex, underscoring the relationship between Determination of macromolecular assembly coatomers and NPCs (Siniossoglou et al., 1996; Salama et al., structures by an integrative approach 1997; Devos et al., 2004; Dokudovskaya et al., 2011). Our approach involves (a) gathering of diverse and complemen- The Nup84 complex is the best characterized of the NPC’s tary experimental data, including data not typically used for building blocks, as re"ected by the extensive set of genetic, bio- structure determination, (b) representation of subunits and transla- chemical, and structural data accumulated over the years (Doye tion of the experimental data into spatial restraints on these sub- and Hurt, 1995; Fabre and Hurt, 1997; Brohawn et al., 2009). units, (c) optimization by satisfaction of the spatial restraints to Mutations of Nup84 complex nups usually lead to severe pheno- produce an ensemble of structures of de!ned precision and con- types characterized by fitness defects, mRNA, and preribo- sistent with the data, and (d) analysis and assessment of the somal export problems as well as aberrant NPC biogenesis and ensemble (Alber et al., 2007a,b, 2008; Lasker et al., 2010). We Downloaded from distribution (i.e., clustering of NPCs into a handful of closely previously demonstrated this approach by determining the yeast packed groups) within the NE; indeed, the NPC clustering pheno- NPC’s molecular architecture based on a diverse set of pro- type has been broadly used as a tool to characterize putative teomic and biophysical data. This map de!nes the approximate NPC-associated proteins (Doye et al., 1994; Aitchison et al., positions of the component proteins but not their shape or ori- 1995; Heath et al., 1995; Li et al., 1995; Pemberton et al., 1995). entations (Alber et al., 2007a,b). We have now signi!cantly en- The Nup84 heptamer forms a characteristic Y-shaped assem- hanced our approach to allow us to easily integrate data spanning bly, as shown by pioneering EM studies of both isolated com- the scale from angstroms to hundreds of nanometers, greatly in- jcb.rupress.org plexes and complexes reconstituted in vitro; Nup133, Nup84, creasing !delity and reliability of our mapping procedure. Here, and Nup145c/Sec13 form the main stalk of the Y, with Nup133 we determine the positions and orientations of the subunits in at its tip, and Nup85/Seh1 and Nup120 are located in the two the yeast Nup84 complex, using data from negative-stain EM, short arms of the heptameric assembly (Siniossoglou et al., af!nity puri!cation and domain mapping, and x-ray structures 2000; Lutzmann et al., 2002; Kampmann and Blobel, 2009). and comparative models of subunits. The advantages of our on March 28, 2012 Structural analyses by combined computational and bio- approach are as follows: (a) precision, accuracy, and complete- chemical methods (Devos et al., 2004, 2006) and subsequent ness of a structure are maximized by bene!ting from the combina- crystallographic studies (Hsia et al., 2007; Boehmer et al., 2008; tion of varied datasets; (b) this precision and accuracy can be Brohawn et al., 2008; Debler et al., 2008; Brohawn and systematically assessed; and (c) ef!ciency is maximized by an Schwartz, 2009; Leksa et al., 2009; Nagy et al., 2009; Seo et al., iterative feedback between data collection and data processing. 2009; Whittle and Schwartz, 2009; Sampathkumar et al., 2011) have shown that nups within the Nup84 complex are formed Gathering data almost entirely by a B-propeller fold, an A-solenoid–like fold We have used the two following primary methods to obtain a (or helix-turn-helix repeat, which from now on we will refer to large quantity of structural information on the Nup84 complex. as A-solenoid), or a combination of N-terminal B-propeller and Domain deletion mapping. Using domain boundary C-terminal A-solenoid–like folds (termed a B-A fold arrange- data (Devos et al., 2004; Dokudovskaya et al., 2006), we expressed ment), again common to VCCs.

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