CAS nuclear retention depends on its N-terminus interaction with RanGTP vis-à-vis SWITCHI. (A) Structure of the yeast homolog of CAS (CSE1; purple) in complex with Kap60 (yeast Kapα; green) and Gsp1pGTP (yeast RanGTP; yellow) (PDB ID: 1wa5). The T18D mutation (red arrow) at the CAS N-terminus is also indicated (see Fig. 7 and main text for details). Its conformation was modeled using the ELASPICwebserver (Witvliet et al., 2016). K67A and N68A mutations (red) on the CAS N terminus and N609A and E656A mutations (black) are shown. (B) Immunostaining of Kapβ1 in cells expressing mCherry-CAS and mCherry-CAS mutants shown. Scale bar, 5 μm. (C) N/C ratios reveal that CAS nuclear accumulation is significantly reduced for mCherry-CAS mutants carrying the K67A and N68A mutations. N608A and E658A mutations in the C-terminus of CAS have no impact on CAS nuclear localization. The dashed line indicates N/C = 1. Box plots denote the median, first, and third quartiles. The whiskers represent the minimum and maximum values. N = 3 or 4. The data were tested for normality using a built-in function of GraphPad Prism software. P values were obtained using an unpaired two-tailed t test. (D) Schematic model illustrating the closed loop of Kapβ1 and CAS that regulates Kapα nuclear import and export, respectively. CAS nuclear retention follows from a “hold” and “release” mechanism at RanGAP1. RanGTP hydrolysis is suppressed by the CAS N-terminus in the “hold” state and achieves “release” by the action of RanBP1 (or RanBP2). See text for details. SWITCH I (I) and SWITCHII (II) on RanGTP are indicated in the cartoon.