1. Volume 18, Issue 3, Pages 355-367 (April 2005)
The Structure of the Nuclear Export Receptor Cse1 in Its Cytosolic State Reveals a Closed Conformation Incompatible with Cargo Binding 
Atlanta Cook, Elena Fernandez, Doris Lindner, Judith Ebert, Gabriel Schlenstedt, Elena Conti 
Molecular Cell 
Volume 18, Issue 3, Pages (April 2005)
DOI: /j.molcel
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2. Figure 1 The Overall Structure of Cse1
The overall structure of Cse1 is shown in two different orientations related by a 180° rotation around the vertical axis. The structure consists of 20 HEAT repeats, numbered H1–H20. The N-terminal half of Cse1 (HEAT repeats 1–8) is colored in blue, with adjacent HEAT repeats in alternating light and dark shades of blue. The C-terminal half of the molecule (HEAT repeats 9–20) is colored in pink, with adjacent HEAT repeats in alternating light and dark color. The insertion at HEAT 8 is highlighted in green. This figure and all others representing structures were generated with the program AESOP (M. Noble, personal communication) unless otherwise stated.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Structure-Based Sequence Alignment of Cse1/CAS with Importin β and Transportin
The alignment includes Cse1 homologs from S. cerevisiae (CSE1_YEAST), S. pombe (CSE1_SCHPO), H. sapiens (CSE1_HUMAN), and D. melanogaster (CSE1_DROME). Highlighted in orange are conserved residues. The secondary structure of Cse1 is shown above the sequence with green rectangles representing α helices. The helices are labeled A and B from HEAT repeats 1–20. Additional interrepeat helices (after HEAT repeats 7, 17, and 18) are shown but not labeled. The intrarepeat helices at HEAT 8 are marked as the insertion. The structure-based alignment with importin β (KAPB1) and transportin (KAPB2) is at the bottom, with the corresponding secondary structure elements previously identified (Chook and Blobel, 1999; Cingolani et al., 1999) boxed in green. The alignment of Cse1 with the importins was created by superposing the N-terminal arches (HEAT repeats 1–7), the C-terminal arches (HEAT repeats 13–20), and the hinge regions (HEAT repeats 7–12) independently with the program DALI (Holm and Sander, 1993).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Conformational Variability of Different Karyopherins
(A) Superposition of cargo-free Cse1 (green) and importin β (gray) at the N-terminal arch (left panel) and at the C-terminal arch (right panel) showing a similar arrangement of HEAT repeats in the two receptors.
(B) Comparison of the different conformations observed in the structures of cargo-free Cse1 (green), importin β (dark gray), transportin (light gray), and cargo-bound Cse1 (yellow). The structures have been superposed at the N-terminal arches and are viewed with the N-terminal arches in a similar orientation as in the left panel of Figure 3A. This figure was created using PYMOL (Delano Scientific).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
The Ring-like Structure of Cse1 Opens up to Assume a Horseshoe Shape upon Cargo Binding
(A) Surface representation of Cse1 in the conformation observed when in complex with RanGTP and importin α. The RanGTP binding sites are shown in blue and importin α binding sites are shown in purple. In the left panel, the molecule is viewed with the C-terminal arch in the same orientation as in Figure 1 (left panel) after optimal superposition while the right panel shows the molecule rotated by 180° about the vertical axis.
(B) Surface representation of unbound Cse1 with the coloring for the binding sites for RanGTP and importin α as in panel (A). The surfaces are orientated as in panel (A).
(C) Surface representation of Cse1 in the unbound state colored according to sequence conservation, ranging from white (variable residues) to dark orange (invariant residues, as in Figure 2).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
The Intramolecular Interaction between the N- and C-Terminal Arches Prevents Cargo Binding in the Absence of RanGTP
(A) A network of interactions between HEAT 1–3 and HEAT 14–16 in the cargo-free conformation. The inset shows the position of the interface according to Figure 1. The interface is mediated by several charged and polar interactions, mainly by conserved residues (see Figure 2).
(B) Pull-down assays with GST-importin α. The Cse1 21,25,28 EEE, Cse1 653R, and Cse1 21,25,28 EEE, 653R mutants are able to interact with importin α in the absence of RanGTP (lanes 7–9). These mutations do not affect the ability of Cse1 to form a cargo-bound complex (lanes 12–15). Mutations to the acidic residues at the HEAT 8 insert region also do not affect complex formation (lanes 17 and 18). In contrast, the Cse1 Δinsert mutant is unable to form a complex with RanGTP and importin α (lane15).
(C) The HEAT 8 insertion is required for cell viability. Yeast cells deleted for chromosomal CSE1 carrying the URA3 plasmid pRS316-CSE1 were transformed with an empty HIS3 vector (−) or a HIS3 plasmid encoding wild-type (WT) Cse1, the Cse1 HEAT 8 Δinsert mutant, the 21,25,28 EEE mutant, or the 653R mutant. The cells were streaked on His- plates or, to counterselect against the URA3 plasmid, on FOA plates and incubated at 30°C for 2 days. The HEAT 8 Δinsert mutant was not viable, however the 21,25,28 EEE and the 653R mutations do not affect cell viability.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
The HEAT 8 Insertion Connects the Importin α and RanGTP Binding Site
Interface between the insertion at HEAT 8 and the B helices of HEAT 9–11 in cargo-free Cse1 (left panel) and cargo-bound Cse1 (right panel, with importin α in purple and RanGTP in blue). Highlighted are the regions of structural changes, with the insertion and helix 8B in green and the loop between HEAT repeats 9 and 10 in orange. The interface comprises extensive ionic and hydrophobic interactions, many of which involve conserved residues (see Figure 2). Charged interactions are indicated with dots. The green dashed line (right panel) indicates the region of the insertion that becomes disordered upon complex formation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions