BIOL2056: Cell Biology Protein Targeting

BIOL2056: Cell Biology Protein Targeting

Recommended Literature

• Alberts, B. Molecular Biology of the Cell, 6th edition, Chapter 12.

• Instructor Contact: Triana Amen, Ph.D. (t.amen@soton.ac.uk)

• Office Location: Building 85, Room 3035

Compartmentalization of Eukaryotic Cells

• Average size of eukaryotic cells: 5 μm.

Learning Objectives

• Why do proteins need targeting pathways?

• How are proteins delivered to different compartments?

• How do we use protein targeting to visualize compartments and assess import mechanisms?

• How does cytoplasm to nucleus protein targeting work?

• Quiz: Identify protein localization signal in a protein sequence.

Importance of Protein Targeting Pathways

• Proteins are produced in the cytoplasm:

  • Eukaryotic cells synthesize proteins primarily in the cytosol.

• Limited diffusion between membrane compartments:

  • Membrane lipid barriers restrict the free passage of proteins and other macromolecules between cellular organelles.

• Essential for organelle functioning:

  • Specific targeting and transport are vital for the proper functioning of organelles (e.g., mitochondria and chloroplasts synthesize a limited number of proteins).

Protein Delivery to Organelles

• Components Required for Protein Transport:

  • Protein Transport Complex

  • Receptor

  • Targeting Sequence

• **Definitions:

  • Protein Targeting Sequence:** A signal that directs the protein to its specific compartment. Also referred to as a protein localization signal.

Machinery Required for Protein Transport

• **Components of Protein Transport:

  • Protein Transport Complex:** Facilitates movement of proteins into organelles.

  • Receptor Protein:* Recognizes the protein targeting sequence.

  • Transport Complex Binds: The receptor-cargo complex is formed which is then imported into the target compartment.

Sorting Signals in Protein Localization

• Example of protein sorting:

  • Cytosolic Protein: Lack of signal sequence.

  • Endoplasmic Reticulum (ER):* ER signal sequences can be attached to cytosolic proteins to target them to the endoplasmic reticulum (ER).

• Illustration of Sorting Signals:

  • ER Signal Sequence Removed and Attached to Cytosolic Protein:

  • Initial states:

    • Cytosolic protein without signal sequence → Protein not transported.

  • ER protein with signal sequence → Transported to ER

    • ER signal sequence removed, protein is either retained or sent to another compartment.

Visualizing Organelles Using Sorting Signals

• Endoplasmic Reticulum (ER): ER localization signal fused to red fluorescent protein (mCherry).

• Peroxisome: Peroxisome targeting sequence (PTS) fused to green fluorescent protein (GFP).

Summary of Protein Targeting Mechanisms

• Questions to be addressed in quizzes include:

  • Name and sequence of protein localization signals.

  • Identification of the protein that binds the targeting signal and delivers cargo to the respective compartment.

  • Determining whether the protein remains folded or unfolds during transport.

  • Timing of protein delivery in relation to translation: co- or post-translational.

Nuclear Targeting

• Nuclear Structure:

  • The nucleus is characterized by a double membrane known as the nuclear envelope.

  • Contains Nuclear Pore Complexes (NPC) for transport of various molecules including proteins and mRNA.

  • Small molecules (<40 kDa) can diffuse freely across the envelope.

Nuclear Pore Complex (NPC)

• Structural Details:

  • The nuclear pore is approximately 50 nm in diameter.

  • Consists of 50-100 proteins referred to as nucleoporins (Nups), including both membrane and peripheral proteins.

  • Central FG-Nups establish a selectively permeable barrier with phenylalanine-glycine motifs to interact with nuclear transport receptors.

  • The NPC functions as a molecular sieve, regulating entry based on macromolecule size.

Nuclear Localization Signals (NLS)

• Classical NLS (cNLS):

  • Characterized by stretches of basic amino acids, specifically lysine (K) and arginine (R).

  • Can be classified as mono- (SV40) or bipartite (e.g., Nucleoplasmin).

  • Example: SV40 large T antigen, sequence: PKKKRKV.

Nuclear Transport Receptors

• Mediated by Importins and Exportins.

  • Importin α: Recognizes and binds to NLS, forming a trimeric complex with importin β.

  • Importin β: Interacts with FG-Nups in the NPC for nuclear entrance.

Ran GTP Cycle

• Involves a conserved protein called Ran necessary for nuclear trafficking.

• Exists in two forms:

  • GTP-bound inside the nucleus.

  • GDP-bound in the cytoplasm.

• The gradient of Ran drives both nuclear import and export mechanisms.

Biochemical Energy Involved in Nuclear Transport

• The hydrolysis of GTP is critical for nuclear transport success.

• When importin α recognizes the NLS, it releases the cargo upon binding with Ran-GTP in the nucleus.

• Following cargo delivery, the Ran-GTP complex with importin is exported back to the cytoplasm, where GTP is hydrolyzed, releasing the receptor for subsequent import.

Summary of Nuclear Targeting Mechanisms

• The NLS can be either mono- or bipartite, consisting largely of basic amino acids (K/R).

• Important considerations in protein localization are:

  • Energy Source: GTP from the RanGTP cycle.

  • NPC: Determines accessibility for macromolecules with some free diffusion allowed for small proteins.

  • Protein State: Proteins must generally be folded at the time of transport and undergo post-translational modifications before entering the nucleus.

Targeting to the Nucleolus

• Example of visualization in human cells shows histone H2B (red) and nucleolar antigen (green).

• The nucleolus plays a critical role in ribosome assembly.

Finding NLS Sequences

• To predict NLS, search for sequences rich in lysine (K) or arginine (R).

• Example sequence for prediction: NPM2 nucleoplasmin, accessible through online tools like NLS Mapper.

Final Summary

• Proteins require localization signals, receptors, and transport complexes for targeting to specific cellular compartments.

• The classical Nuclear Localization Signal (NLS) involves basic amino acids either in mono- or bipartite arrangements.

• Targeting of proteins from the cytoplasm to the nucleus is energy-dependent and facilitated by the NPC, which is a complex molecular apparatus.

Recommended Literature and Open Questions

• Further Reading:

  • Alberts, Molecular Biology of the Cell, 6th ed., Chapter 12.

  • Miyamoto et al., "Protein transport between the nucleus and cytoplasm," 10.1016/B978-0-12-803480-4.00025-9.

  • Dingwall and Laskey, 1998. "Nuclear Import: a tale of two sites." 10.1016/s0960-9822(98)00010-4.

• Potential Research Questions:

  • How do cells export proteins from the nucleus using exportins and nuclear export signals (NES)?

  • What is the cutoff size for molecules diffusing into the nucleus?

  • What are the mechanisms for nuclear pore complex assembly?

  • How are NPCs affected during cell division?

Terminology to Understand

• Protein, amino acids, protein folding, protein translation, protein localization sequence, nuclear pores, importin/karyopherin.

Compartmentalization and Protein Sorting

The Necessity of Compartmentalization

• Eukaryotic cells are subdivided into functionally distinct, membrane-enclosed compartments. On average, these cells measure approximately $5 \mu m$.

• Specialized Environments: Compartmentalization allows for different chemical environments (e.g., low pH in lysosomes) to coexist within the same cell, optimizing enzyme activity and metabolic pathways.

• Barriers to Diffusion: The lipid bilayer prevents the free passage of hydrophilic proteins and macromolecules. Consequently, specific active transport mechanisms are required to move proteins from their site of synthesis (cytoplasm) to their functional destination (organelles).

Fundamental Sorting Mechanisms

1. Gated Transport: Movement between the cytosol and nucleus through Nuclear Pore Complexes (NPCs). These act as selective gates that support the active transport of specific macromolecules while allowing free diffusion of small molecules.

2. Transmembrane Transport: Protein translocators move proteins across a membrane from the cytosol into a space that is topologically distinct (e.g., ER, mitochondria). This usually requires the protein to unfold.

3. Vesicular Transport: Membrane-enclosed transport intermediates (vesicles) ferry proteins from one compartment to another (e.g., ER to Golgi).

Components of Protein Targeting

Sorting Signals

• Signal Sequences: Continuous stretches of amino acids (typically 15-60 residues long) that direct proteins. Once the sorting process is complete, these sequences are often removed by signal peptidases.

• Signal Patches: Formed by the three-dimensional arrangement of amino acids on the protein's surface when it folds. These are common for nuclear targeting and lysosomal sorting.

• Localization Examples:

  • ER Targeting: Often involves a hydrophobic sequence at the N-terminus.

  • Nuclear Targeting (NLS): Rich in positively charged (basic) residues like Lysine ($K$) and Arginine ($R$).

Delivery Machinery

• Receptors: Specialized proteins (e.g., Importins) that recognize and bind specifically to sorting signals.

• Translocons/Transport Complexes: Proteinaceous channels or pores that facilitate the physical translocation of the cargo across the lipid bilayer.

Nuclear Pore Complex (NPC) Structure and Function

• Physical Scale: The NPC is a massive structure (~$125 \text{ MDa}$ in vertebrates) composed of approximately 30 different proteins called nucleoporins (Nups).

• Structural Components:

  • Scaffold Nups: Form the core ring structure and anchor the complex to the nuclear envelope.

  • Channel Nups (FG-Nups): Line the central pore. They contain disordered regions rich in Phenylalanine-Glycine (FG) repeats. These repeats create a "hydrophobic mesh" or sieve that prevents the passage of macromolecules larger than $40 \text{ kDa}$ while allowing small molecules to diffuse.

• Selectivity: To bypass this mesh, large proteins must be bound to nuclear transport receptors that interact transiently with the FG repeats to "dissolve" through the barrier.

The Ran GTPase Cycle: The Engine of Nuclear Transport

Nuclear transport is an active, energy-dependent process fueled by the Ran GTPase gradient.

Two States of Ran:

1. Ran-GTP: Found in high concentrations in the nucleus. This is maintained by Ran-GEF (Guanine nucleotide Exchange Factor), which is sequestered in the nucleus by binding to chromatin.

2. Ran-GDP: Found in high concentrations in the cytosol. This is maintained by Ran-GAP (GTPase Activating Protein), which is located in the cytoplasm.

Transport Mechanisms:

• Nuclear Import:

  1. Importin binds the NLS-containing protein in the cytosol.

  2. The complex moves through the NPC.

  3. Inside the nucleus, Ran-GTP binds to Importin, causing it to release the cargo.

  4. The Importin-Ran-GTP complex exports back to the cytosol.

  5. Ran-GAP induces GTP hydrolysis; Importin releases Ran-GDP and is ready for a new cycle.

• Nuclear Export:

  1. Exportin binds both the cargo (containing a Nuclear Export Signal or NES) and Ran-GTP simultaneously in the nucleus.

  2. The trimeric complex moves to the cytosol.

  3. Ran-GAP induces hydrolysis ($\text{GTP} \rightarrow \text{GDP}$), triggering the disassembly of the complex and release of the cargo into the cytoplasm.

Clinical and Research Applications

• Visualizing Compartments: Researchers use fusions of fluorescent proteins (like GFP or mCherry) with specific targeting signals (e.g., mCherry-KDEL for ER or GFP-NLS for nucleus) to observe organelle dynamics in live cells.

• Pathology: Mutations in Nups or NLS sequences are linked to various diseases, including certain types of leukemia and neurodegenerative disorders where protein nucleocytoplasmic transport is impaired.

Quantitative Considerations:

• Diffusion Limit: Molecules < $9 \text{ nm}$ in diameter or < $40 \text{ kDa}$ can typically pass by passive diffusion.

• Active Transport Gate: The NPC can expand to accommodate complexes up to approximately $26-39 \text{ nm}$ in diameter.