Nuclear import

1. Nuclear import

2. Translocation through the nuclear pore free diffusion assisted transport nuclear proteins ribosomal proteins small molecules (<50 kD) tRNAs mRNAs ribosomal subunits

3. Cytoplasmic side Xenopus nuclear pore complex Nuclear side (T. Allen )

4. Vertebrate nuclear pore complex (NPC) (C. Akey) cytoplasmic filaments basket cytosol nuclear membranes nucleus

5. Nuclear Targeting Sequences Import:Nuclear Localization Sequence (NLS) - Classical or basic NLS - M9 NLS found on hnRNP A1 Export:Nuclear Export Sequence (NES) - Leucine-rich NES - tRNA NES (intrinsic to tRNA, not associated proteins)

6. Assay for nuclear import with digitonin-permeabilized cells Treat tissue culture cells with low digitonin (digitonin permeabilizes the plasma membrane while leaving the nuclear envelope intact) Add nuclear import substrate (TRITC-NLS-BSA) Detect nuclear protein import by fluorescence Add cytosol and energy

7. To test requirements for binding to the nucleus Incubate digitonin-permeabilized cells with TRITC-NLS-BSA TRITC-NLS-BSA alone + cytosol Conclusion: Cargo does not interact directly with the NPC. Instead, it needs a carrier.

8. Karyopherin b family members transport multiple classes of cargo Kap95p Kap60p-NLS substrates Kap104p mRNA binding proteins (Nab2p, Hrp1p) Kap111p /Mtr10p mRNA binding protein (Npl3p) Kap123p ribosomal proteins Kap108p /Sxm1p La, ribosomal proteins Kap121p /Pse1p transcription factor, ribosomal proteins Kap119p /Nmd5p transcription elongation factor (TFIIS), kinase Kap114p general transcription factor (TBP) Kap122p /Pdr6p general transcription factor (TFIIA) IMPORT EXPORT Kap124p /Crm1p NES substrates Kap109p /Cse1p Kap (Kap60p) Kap127p /Los1p tRNA BOTH Kap142p/ Msn5p Import: trimeric replication protein A (RPA) Export: transcription factor (Pho4p), Far1p,

9. To identify proteins in the nuclear pore complex that bind nuclear carriers: ? Separate nuclear envelope proteins by SDS-PAGE. Blot to nitrocellulose Incubate the blot with NLS-BSA, Kap-a and Kap-b1. Detect binding with anti- BSA antibody

10. Nup358 Nup214 Nup153 Nup98 Nup62 NLS-BSA binds to a number of nuclear envelope proteins Abundant Repeat-Containing Nucleoporins (FG, FXFG, GLFG) line the route through the nuclear pore Nup358 Nup214 Nup62 Nup98 Nup153 from Radu, Blobel, and Moore, 1995

11. Is the movement of a carrier from one NPC site to the next... directional? or random? Answer is not yet known

12. Yeast FG Nucleoporins (FG Nups) NUP192 NUP188 NUP170 NUP159 NUP157 POM152 NUP145 NUP133 NUP120 NUP116 NUP100 NUP1 NIC96 NSP1 NUP85 NUP84 NUP82 GLE1 GLE2 NDC1 NUP60 NUP59 NUP57 NUP53 NUP49 NUP42 POM34 SEH1 SEC13 CDC31

14. Ran = Ras related nuclear protein A 25 kD GTPase that is extremely abundant and highly conserved from plants to man Present in every nucleated eukaryotic cell tested thus far (probably every nucleated cell on earth)

15. Import of TRITC-NLS-BSA into the nuclei of digitonin permeabilized cells TRITC-NLS-BSA + carriers + Ran + GTP TRITC-NLS-BSA + carriers (carriers = Kap-b1 and Kap-a) TRITC-NLS-BSA alone Conclusion: Ran + GTP is required for nuclear import of proteins with NLS (this is how Ran was originally identified -adapted from Moore and Blobel, 1993 )

16. How do import and export complexes know when to disassemble? cargo carrier - NPC allows free diffusion <40-50 kDa - pH of cytoplasm and nucleus are identical - ionic composition identical - reducing potential identical - electrical potential identical - no post-translational modifications cytoplasm nucleus Instead, transport complexes of the Kap-b family use the concentration of RanGTP as a positional cue to regulate their assembly and disassembly. cargo carrier

17. Transport and the Ran Cycle Import Complex Assembles in presence of GDP-Ran Disassembles in presence of GTP-Ran Export Complex Disassembles in presence of GDP-Ran Assembles in presence of GTP-Ran cytosol nuclear membrane nucleus

18. Ran-GTP and Ran-GDP form gradients across the NPC Ran-GDP GAP Pi GAP Ran-GDP RanGTP Ran-GDP Ran-GDP GAP GAP cytoplasm nucleus GDP GTP Ran-GTP Ran-GTP Ran-GDP Ran-GTP Ran-GTP GEF Ran-GTP GEF GEF

19. All Kap-b carriers bind RanGTP but not RanGDP. Binding of RanGTP has opposite affects on the ability of import versus export carriers to simultaneously bind cargo. Import carriers release their cargo in the nucleus upon binding RanGTP. Export carriers require bound RanGTP to simultaneously bind cargo with high affinity (nucleus). Upon contact with the RanGAP in the cytoplasm, Ran hydrolyzes its bound GTP (forming RanGDP) and the export complex dissociates.