Unit 03 pt5

Overview of Protein Sorting into the Nucleus

• Focus: Understand the transportation of proteins between the cytoplasm and the nucleus, specifically through the nuclear pore complexes.

Structure of the Nucleus

• Nuclear Envelope:

  • Encloses DNA, defining the nuclear compartment.

  • Composed of two concentric membranes (inner and outer).

  • Nuclear Pore Complexes (NPCs) penetrate the nuclear envelope.

• Inner Nuclear Membrane:

  • Contains specific proteins as anchoring sites for the nuclear lamina.

  • Provides structural support and anchors chromatin.

• Outer Nuclear Membrane:

  • Continuous with the Endoplasmic Reticulum (ER).

  • Ribosomes are attached, allowing protein synthesis that can be directed to the perinuclear space.

• Perinuclear Space:

  • Space between inner and outer membranes, continuous with the ER lumen.

Transport Mechanisms

• Bidirectional Transport through NPCs:

  • Movement of molecules between the cytosol and the nucleus.

  • Includes import of nuclear proteins (e.g., histones, RNA polymerases) and export of tRNAs, mRNAs.

  • Only mature mRNAs are exported.

• Nuclear Pore Complex (NPC):

  • Composed of about 30 proteins (nucleoporins), about 125 megadaltons in size.

  • Symmetric structure: 8-fold rotational and 2-fold transverse.

  • Thousands of NPCs in a typical mammalian cell, each capable of transporting about 1,000 macromolecules per second.

Gated Transport

• Gated Transport Definition:

  • Only molecules below a certain size can diffuse freely through NPCs.

  • < 5 kilodaltons can freely diffuse; > 16 kilodaltons find entry difficult.

  • Molecules in between can diffuse but at a slower rate.

• Molecular Sieve Mechanism:

  • Certain size-selective transport characteristics; larger molecules require active transport.

Nuclear Localization Signal (NLS)

• Importance of NLS:

  • Essential for selective import into the nucleus.

  • Typically 1-2 amino acids long, rich in lysines and arginines.

• Experimental Determination:

  • Mutation analysis shows that the integrity of the NLS is important for nuclear import.

• Nuclear Import Receptor (Importin):

  • Binds to NLS for transport into the nucleus.

  • Can be conjugated to other proteins to facilitate nuclear transport.

  • Large protein complexes may only need one subunit with NLS.

FG Repeats and Import Mechanism

• FG Repeats:

  • Found on NPCs, rich in phenylalanine (F) and glycine (G); assist in receptor binding.

  • Repeated binding and dissociation facilitate cargo transport.

• Cargo Release:

  • Once in the nucleus, importins release their cargo due to binding with Ran GTP.

Nuclear Export Mechanism

• Exportins:

  • Nuclear export receptors that bind Ran GTP and cargo for export from the nucleus.

  • Return to the nucleus afterward.

• Cargo and Ran GTP Gradient:

  • The gradient of Ran GTP in the nucleus drives the directionality of transport.

Ran Protein and Energy Requirement

• Ran Protein:

  • A RAS-related nuclear protein, crucial for nuclear transport.

  • Exists in GTP-bound (active) and GDP-bound (inactive) forms, with transport utilizing GTP hydrolysis.

• Function of Ran GTP:

  • Maintains gradient necessary for import/export processes.

  • Ran GTP is high in the nucleus, promoting cargo binding; Ran GDP is found in the cytosol.

Molecular Switches

• Protein Regulation Examples:

  • Phosphorylation through protein kinases can activate or inactivate proteins.

  • GTP-GDP Binding States:

    • GTP-bound state usually indicates active protein; GDP-bound state typically signifies inactivity.

Regulation of Nuclear Import/Export

• Factors Influencing Transport:

  • Post-translational modifications can affect nuclear localization, represented by SREBP protein regulation.

Mitosis and Nuclear Envelope Breakdown

• Nuclear Envelope Breakdown During Mitosis:

  • Inner nuclear envelope structural changes result from phosphorylation of nuclear lamins.

  • Ran gradient maintained despite nuclear envelope breakdown due to anchoring of Ran gap to chromatin.

Summary

• Understanding protein transport dynamics into and out of the nucleus is crucial for central cellular processes such as gene expression and cellular regulation.