The yeast nuclear pore complex and transport through it

Abstract

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell's genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or "Nups"), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC's role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins.

Figure 1

Visualizing the yeast NPC. (A) Transmission EM transverse sections of the NE revealing cytoplasmic filamants (large arrows), nuclear baskets (arrowheads), and interbasket connections (small arrows) (Rout and Blobel 1993). (B) Scanning EM showing a bird’s eye view of cytoplasmic filaments (top, arrow) and the nuclear basket (bottom, arrowhead) (Kiseleva et al. 2004). (C) En face slice of the mass density distribution from a cryoEM map of the yeast NPC (Yang et al. 1998). Two equivalent peaks per spoke unit are seen for the outer rings (1–3) and the inner rings (2–4) (see Figure 2). (D) En face surface-rendered view from a cryoEM map of the yeast NPC (Yang et al. 1998). (E) Projections of Nup mass density, derived from the combined Nup localization volumes (Alber et al. 2007b). (F) The structured nucleoporin domains of the NPC, represented by a density contour approximated to the combined volume of the 456 nucleoporins composing the NPC (Alber et al. 2007b).

Figure 2

(A) Major structural features of the yeast NPC (based on the architectural map of Alber et al. 2007a,b); see main text for details. (B) Map of protein positions in the yeast NPC (based on the architectural map of Alber et al. 2007a,b), with examples of the atomic structures of pieces of Nups where known: Nic96 (2RFO) (Schrader et al. 2008), Nup84/Nup145C/Sec13 (3IKO) (Nagy et al. 2009), Nup85/Seh1 (3EWE) (Brohawn et al. 2008), Nup116 (2AIV) (Robinson et al. 2005), Nup120 (3F7F) (Seo et al. 2009), Nup133 (3KFO), Nup145N (3KEP) (Sampathkumar et al. 2010), Nup159 (1XIP) (Weirich et al. 2004), and Nup170 (3I5P) (Whittle and Schwartz 2009).

Figure 3

Map of major fold-type positions in the yeast nucleoporins [adapted from Devos et al. (2006)]. Here, for the sake of clarity, we define Nups as proteins that appear stoichiometrically and stably associated with the NPC. The sequence of each yeast Nup is represented to scale as a thin black horizontal line. Predicted transmembrane helices are shown in dark green, cadherin domains are in dark blue, coiled coils and α-helical coils are shown in red. β-Propellers are shown in cyan, α-solenoid domains are in magenta, the autoproteolytic domain is in yellow, and the RRM is shown in orange. Unstructured regions are shown by an empty box, except for the FG-repeat regions, which are colored blue-green for low-DERK (Asp, Glu, Arg, Lys) regions and light green for high-DERK regions; the position of each FG repeat is shown as a short green vertical line below each horizontal black sequence line. Representative models are shown on the left of the Nup domains and are colored according to the fold type. Dark-gray bars below each horizontal sequence line mark the position of crystal structures solved for yeast Nups (e.g., N-terminal region of Nup159). Position of folds is based on Devos et al. (2004, 2006); and position and type of FG repeat is based on Yamada et al. (2010).

Figure 4

The nuclear transport cycle for karyopherins and their cargoes. See Fig. 5 legend and main text for details.

Figure 5

The transport cycle of Kap60 and Kap95 is shown diagrammatically in the center, with relevant atomic structures shown in the surroundings. (A) The extended NLS attached to a GFP reporter [green; PDB 1EMA (Ormo et al. 1996)] binds to a long region on the inside of the Kap60 superhelix [dark blue; PDB 1EE5 (Liker et al. 2000)], made of alternating α-helical turns. (B) The characteristic superhelical solenoid of Kap95 (light blue), made of alternating α-helical turns in a related fashion to Kap60, forms a spiral with two surfaces. The inner surface wraps around the extended N-terminal IBB domain of Kap60, which links it tightly to Kap95 [PDB 1QGK (Cingolani et al. 1999)]. (C) As Kap95 passes through the NPC, it interacts with FG Nups. The repeated Phe residues on the FG-repeat region (red) insert into complementary repeated pockets formed from the crevices between adjacent α-helical repeats, all along the outer surface of Kap95’s spiral [PDB 2BPT (Liu and Stewart 2005)]. By transferring between the multiple FG repeats in the NPC, Kap95—together with Kap60 and its NLS-GFP cargo—cross the NPC. (D) In the nucleus, binding of RanGTP (orange) to Kap95 [PDB 2BKU (Lee et al. 2005)] causes a conformational change in the latter, which releases Kap60, and, in doing so, Kap60 is made to release its NLS cargo into the nucleoplasm. In either its Ran bound or free form, Kap95 can bind to FG Nups and thereby cross the NPC to continue the transport cycle. (E) Kap60 is exported from the nucleus by the RanGTP-bound form of the karyopherin Cse1 [magenta; PDB 1WA5 (Matsuura and Stewart 2004)]. In this state, the IBB domain is held tightly against the side of Kap60, inhibiting NLS binding. Both Kap60 and RanGTP are once again held to the inner surface of the Cse1 spiral, leaving the outer surface free to interact with FG repeats and carry the complex through the NPC out of the nucleus. Once in the cytoplasm, GTP on Ran is hydrolyzed to form RanGDP, causing the complex to dissociate. Kap60 remains bound to its IBB even when free in the cytoplasm, but binding to an NLS exposes the IBB and allows Kap95 to bind, initializing another round of import. (F) As a result of the import cycle, NLS-GFP accumulates in the nucleus over time, shown here by fluorescence microscopy (Timney et al. 2006).

Figure 6

Diagrammatic representation of mRNA export, adapted from Strambio-de-Castillia et al. (2010). The SAGA complex is recruited to the promoter of a subset of inducible genes and promotes their transcription. SAGA and the NPC-associated TREX-2 complex may help the genes move to the vicinity of the NPC. The nascent transcripts recruit shuttling mRNA-coating factors, THO, TREX, and, subsequently, the mRNA export factors Mex67p and Mtr2p, resulting in the formation of an export-competent mRNP (Rodriguez-Navarro and Hurt 2011); the association of the maturing mRNPs with components of the nuclear basket is strengthened in preparation for nuclear translocation, while nuclear basket-associated TRAMP and exosome complex-associated mRNP surveillance mechanisms ensure that the mRNP is correctly assembled for export (Fasken and Corbett 2009). After translocation through the NPC, the release of mRNA export factors from mRNPs is induced by the combined action of Dbp5p and Gle1p, which are docked to NPC cytoplasmic filaments via interaction with Nup42p and Nup159p, respectively, and are thought to act as mRNP-remodelling factors (Carmody and Wente 2009). It is presumed that this process drives the directionality of mRNP export while at the same time priming mRNAs for translation initiation.