Translocation

• Principles of protein targeting to compartments:

  • Sorting signals of polypeptides
  • Folding and unfolding events
  • Recognition factors for sorting signals
  • Translocation machinery
  • Energy required
  • Processing
• Some signals are in the N terminal and consist of contiguous sequences, others are internal and non contiguous sequences
• Nucleocytoplasmic transport can be via gated transport or transport channels
• Gated transport:

  • Continuous bi directional transport between nucleus and cytosol
  • Selective requires receptor / cargo complex regulated transport
• Transport channel:

  • Nuclear pore complex - located in nuclear membrane
  • 125 mega Da
  • Small proteins (5 kDa) - passive diffusion
  • Large proteins (more than 30 kDa and 5 nm diameter) require receptor
  • Transport of fully folded proteins
• Nucleocytoplasmic transport requires a cargo signal - nuclear localisation signals (NLS)
• Nucleocytoplasmic transport requires cargo receptors - karyopherins (importins and exportins)
• Amphiphatic - having polar and non polar structure
• Nuclear localisation signals (NLS) are often formed from separate residues often rich in lysine and arginine at any position of a protein
• Only one subunit of a multisubunit protein complex requires an NSL for import
• Proteins may contain nuclear export signals (NES) in addition to NLS
• Nuclear localisation signals (NLS) on cargo bind cytosolic nuclear import receptors (importins)

  Each receptor binds a subset of cargo proteins (NLS determined) and nuclear pore complex (NPC) fibril proteins
• Some cargos require adaptors to bind to import receptors
• Receptor-cargo complexes cross the pore by a series of weak, transient interactions of hydrophobic surface pockets of importin beta with FG repeats in NPC fibril proteins
• FG repeats form hydrogel meshwork which controls transport of receptor-cargo complexes
• Import of nuclear proteins is an energy dependent processes - energy is obtained from GTP hydrolysis by the monomeric G-protein RanGTPase
• Ran (RanGTP in nucleus and RanGDP in cytosol) is a molecular switch with two confirmations that requires:

  • Cytosolic GTPase Activating Protein (Ran-GAP)
  • Nuclear Guanine nucleotide Exchange Factor (Ran-GEF) which promotes exchange of GDP for GTP
• Nuclear import

  
  

  Cytosolic side

  1 Importin + cargo / NSL bind to NPC

  2. FG repeat binds through the nuclear pore

  
  

  Nuclear side

  3. RanGTP binds (promoted by RanGEF) which releases the cargo into the nucleus

  4. Receptor-RanGTP is transported to the cytosol

  
  

  Back to cytosolic side

  5. RanGAP promotes GTP hydrolysis

  6. RanGDP is released from the receptor
• Nuclear export

  Nuclear side

  1 Exportin + cargo / NES binding is promoted by RanGTP

  2. RanGTP cargo receptor passes through NPC

  Cytosolic side

  3. RanGAP promotes GTP hydrolysis

  4. Cargo + exportin released

  5. Free receptor is transported into the nucleus

  Back to nuclear side

  6. RanGDP that was imported into nucleus binds to its own receptor
• The direction of distribution in and out of the nucleus is driven by the distribution of RanGAP and RanGEF:

  • RanGEF in nucleus drives release of cargo with NLS in nucleus
  • RanGAP in cytosol drives release of cargo with NES
  • Post translational SUMO (small ubiquitin like modifier) lation of RanGAP - allows its association with the cytosolic face of the nuclear pore