MCB 2210 L 13: Protein Targeting: Nucleus Part 2

What protein allow the Nuclear Localization Signal (NLS) and the Nuclear Export Signal (NS) to be recognized? + 2 types + protein that regulates process

• Karyopherins = family of transport proteins that move molecules in/out of nucleus THROUGH nuclear pore complex

  • Importin = nuclear import receptors = help proteins into nucleus

  • Exportin = nuclear export receptors = help proteins out of nucleus

• Ran = small G-protein that regulates both processes

• Importin & exportin = of same gene family (related sequences = of same gene family)

  • Function similar BUT opposite directions

Describe G protein cycle

1. Ran + GDP = INACTIVE

2. GEF replaces GDP for GTP

3. Ran + GTP = ACTIVE

4. GAP cleaves phosphate group

   1. GTP → GDP

5. Returns Ran to inactive state

What are 2 types of RAN regulator proteins used to control nuclear transport? How do they work together to regulate nuclear transport? What drives transport?

RanGAP = enzyme that turns Ran OFF

• Located exclusively in cytoplasm

• Cleaves P_i off of GTP → GDP

• Ran-GDP = ONLY in cytoplasm

RanGEF = enzyme that turns Ran ON

• Located exclusively in nucleus (bound to chromatin)

• Exchanges GDP ↔ GTP

• Ran-GTP = ONLY in nucleus

RanGAP & RanGEF = nuclear/cytoplasmic switch to import/export proteins to drive directional transport

• Asymmetrically located → proteins “segregated” into nucleus & cytoplasm

GTP → hydrolyzed by GAP (GTP → GDP) = energy needed for transport

• Anentropic process = create order by forcing molecules into nucleus (opposite entropy)

  • Used to reset the passive transport of molecules through Nuclear Pore Complex (NPC)

  • Importin + molecule → NPC into nucleus

  • Importin + Ran-GTP → LETS GO of molecule in nucleus

  • Importin + Ran-GTP → OUT to cytoplasm

    • RanGAP → Ran-GTP → Ran-GDP + P_i

      • Ran Δ shape → lets go of importin & grab new molecules

Describe the Nuclear Import Cycle pathway

1. Importin = nuclear import receptor → recognizes Nuclear Localization Signal (NLS) → binds to cargo molecule to be imported

2. Importin + cargo binds to Nuclear Pore

3. Importin + cargo transported through pore into nucleus

4. Importin + cargo encounter Ran-GTP in nucleus

5. Importin + cargo bounded to by Ran-GTP → cargo released

6. Importin + Ran-GTP → cytoplasm

7. Importin + Ran-GTP encounter RanGAP → Ran-GTP → Ran-GDP

8. Importin - Ran-GDP (released)

9. Ran-GDP → nucleus by NTF2 (specific import receptor)

   1. Nuclear Transport Factor 2 = cytosolic protein → imports Ran-GDP from cytoplasm → nucleus

Describe the Nuclear Export Cycle Pathway

1. Exportin = nuclear export receptor → recognizes Nuclear Export Signal (NES) + cargo molecule

2. Exportin + cargo (tripartite complex) bounded by Ran-GTP → ACTIVATES export

3. Exportin + cargo (tripartite complex) encounters cytoplasmic RanGAP

   1. RanGTP → RanGDP

4. Exportin + cargo (tripartite complex) + RanGDP → DISSOCIATE

5. Cargo = delivered

6. Exportin returns to nucleus

7. Ran-GDP → nucleus by NTF2 (specific import receptor)

   1. Nuclear Transport Factor 2 = cytosolic protein → imports Ran-GDP from cytoplasm → nucleus

What does Ran use to be selectively reimported into nucleus and why?

Ran uses Nuclear Transport Factor 2 (NTF2) for Ran-GDP to be brough through nuclear pore complex (NPC)

• DOES NOT HAVE Nuclear Localization Signal nor Nuclear Export Signal

Describe the 4 different pathways that allow different components of nuclear transport are recycled

1. Recycling of Ran-GDP

   1. PROBLEM: Ran = constantly exported to cytoplasm

   2. SOLUTION: Ran-GDP returned to nucleus by NTF2 → Ran-GEF converts Ran-GDP → Ran-GTP (active)

2. Nuclear Import = protein → nucleus (from cytoplasm)

   1. Cargo = protein w/ Nuclear Localization Sequence (NLS)

   2. Importin + NLS of cargo → protein through pore → nucleus

   3. Importin + cargo bound by Ran-GTP → cargo released in nucleus

3. Recycling of Importin-α

   1. CAS (specialized export receptor) → binds to Important + Ran-GTP

   2. Nucleus → cytoplasm → Ran-GTP hydrolyzed → Ran-GDP → Importin = released

4. Nuclear Export = protein → cytoplasm (out of nucleus)

   1. Cargo = protein w/ Nuclear Export Sequence (NES)

   2. Exportin + NES of cargo + Ran-GTP → cytoplasm

   3. Ran-GAP breaks down Ran-GTP → complex dissociates → cargo released in cytoplasm

What is required for transport through nuclear pore complex (NPC)?

Phenylalanine-glycine (FG) repeats found in nucleoporins = form mesh-like barrier inside nuclear pore complex (NPC)

• Barrier = selective gate for transporting molecules in & out of nucleus

• Mechanism = UNKNOWN

How are the majority of RNA species (including ribonuclear proteins) exported from nucleus?

Nuclear pores

• Interact w/ exportins to get through

What is the relationship between types of exportins & types of RNAs?

Experiments demonstrate that there are different types of exportins for different kinds of RNAs

How is a ribosomal subunits formed in the nucleus?

1. Ribosomal proteins → nucleolus through NPC

2. Ribosomal proteins + pre-rRNA

3. Eventually bind to Ran-GTP → brought to cytoplasm through NPC

   1. Ran-GTP → Ran-GDP → subunits formed

How is transport of proteins across nuclear envelope regulated?

• Protein + targeting signal ≠ always moving through pore

  • May be bound to other structure = immobilized

  • Signal may be buried in protein (wrong conformation)

  • Signal may be concealed by another protein

• Upstream signal (hormone/chemical Δ) → Δ conformation → expose signal → allow protein to be imported

What are the 2 examples of nuclear transport regulation?

1. NF-AT and T-Cells

2. Steroid hormones + Nuclear Hormone Receptor

Explain the transcription factor import is regulated in T-Cell Activation (T-cells + NF-AT)

• ON

  • NF-AT in cytoplasm of resting T-cell = phosphorylated → hides NLS

    • NF-AT = family of five transcription factors = regulates gene expression

  • Signaling → Ca²⁺ levels INCREASE

  • Calcineurin (Ca²⁺ dependent phosphatase) binds to NF-AT

  • NF-AT - Phosphate → NLS = EXPOSED

  • Importin recognizes NLS → carries NF-AT → nucleus

  • Transcription = ON

• OFF

  • Ca²⁺ levels DECREASE → Calcineurin RELEASES NF-AT

  • NF-AT + Phosphate → NLS = hidden, NES = EXPOSED

  • Exportin recognizes NES → kicks NF-AT back to cytoplasm

  • Ensure immune response = off

How do steroid hormones affect gene expression?

• Steroid hormones = nonpolar → diffuse through membrane

  • Hormone binds to Nuclear Hormone Receptor in cytoplasm

  • Δ conformation of Nuclear Hormone Receptor → expose/activate NLS (import signal)

  • Importin grabs hormone-receptor complex → imported to nucleus

Describe how nuclear proteins are re-recruited to the nucleus during mitosis

• Nuclear membrane & lamina break down

• Nuclear pore complexes (NPCs) break down into subcomplexes

• Mitotic spindle forms

• Chromosomes segregate (karyokinesis) → nucleus reassembles around DNA

  • Nuclear proteins re-recruited to nucleus through NLS

  • Unclear how NPC reassembles

• Cell membrane & cytoplasm divides (Cytokinesis)