Lecture 9: The Nuclear Pore

Compartmentalisation of the cell increases efficiency and allows for specialisation. This is exemplified by structures such as the expansion of the Endoplasmic Reticulum (ER).
Intracellular protein transport occurs across various compartments including the cytosol, nucleus, plastids, mitochondria, peroxisomes, ER, late endosome, lysosome, Golgi, early endosome, and secretory vesicles.
There are three main types of transport mechanisms:
- Gated transport: Examples include the movement of proteins between the cytosol and the nucleus via the nuclear pore.
- Transmembrane transport: Directly from the site of synthesis to a destination organelle.
- Vesicular transport: Encapsulation of proteins in vesicles for movement between compartments.
Sorting signals are required for proper protein delivery. These act as an "address label" to ensure the protein reaches its intended location.

The nuclear pore is formed at the junction of the inner and outer membranes of the nuclear envelope.
The nuclear pore complex is not a simple hole; it consists of multiple copies of approximately 30 different nucleoporins.
Each complex is composed of 8 subunits with a central plug.
Function of nuclear pore complexes:
- They transport substances into and out of the nucleus.
- During DNA synthesis, histone molecules produced in the cytoplasm are transported into the nucleus to package new DNA.
- Ribosomal subunits ( $60s$ and $40s$ ) formed in the nucleolus must be exported to the cytoplasm for protein production to occur.
Advantages of separating transcription and translation:
- Quality Control: mRNA can be processed through capping and splicing before translation.
- Efficiency: Allows for the repeated use of a single mRNA molecule.
- Increased Complexity: It allows for separate rates of transcription and translation. The transcriptome (the mRNA present) and the proteome (the proteins present) can overlap but are not identical.

Transport through the pore complex occurs via two methods:
- Diffusion: Small molecules move freely. Molecules up to $5000$ daltons are freely diffusible. Molecules of $17,000$ daltons take approximately 2 minutes to reach equilibrium. Molecules of $44,000$ daltons take 30 minutes to reach equilibrium. Proteins weighing $60,000$ daltons or more cannot enter by diffusion.
- Active Transport: Required for larger molecules. Under appropriate signaling, the pore can open to a diameter of up to $26\,nm$ .
Nuclear transport signals are linked to specific peptide sequences known as nuclear transport recognition sites, which are characteristically rich in Lysine (Lys), Arginine (Arg), and Proline (Pro).
The T antigen of the SV40 virus is a standard example of such a signal, containing the sequence: Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val. Disruption of this sequence prevents localization to the nucleus.
Evidence for active transport:
- In cells, the transport of RNA is inhibited at $4^{\circ}C$ .
- In vitro experiments show that ATP is required for fluorescently labeled proteins to enter the nucleus. Without ATP, proteins bind to the nuclear pore but remain outside.

The ER is a central hub for protein synthesis, creating membrane proteins for the lysosome, plasma membrane, and mitochondria, as well as secreted messages like growth factors and morphogens.
The Signal Hypothesis, for which Gunther Blobel won the Nobel Prize, posits that a specific signal sequence is required for translocation.
Translocation can be co-translational or post-translational:
- Co-translational translocation: The most common entry into the ER. As the protein is synthesized by a ribosome bound to the rough ER membrane, it is fed into the ER lumen. Ribosomes are held tightly to the membrane rather than being free in the cytosol.
- Post-translational translocation: Occurs after the polypeptide chain is fully synthesized; this is also common in organelles like mitochondria and chloroplasts.
The ER environment is distinct from the cytosol and must be maintained. The translocator pore acts like a "doughnut" that can open and close to facilitate threading the polypeptide while maintaining this environment.
Signal peptidase is an enzyme located in the ER that cleaves the signal sequence from the mature polypeptide chain once it has entered the lumen.

Single-pass transmembrane proteins are inserted using start-transfer and stop-transfer sequences:
- A hydrophobic stop-transfer sequence anchors the protein in the membrane.
- The translocator pore opens laterally to release the signal sequence into the lipid bilayer.
Type II topology for single-pass proteins features the N-terminus in the cytosol. In this case, the signal sequence is located midway through the polypeptide rather than at the end and remains part of the final membrane structure.
Chaperones, such as BiP, ensure proper protein folding within the ER.
- BiP associates with heavy and light chains of proteins (like antibodies) as they are fed through the translocator.
- Proteins are retained in the ER until they are folded correctly; only properly folded proteins are allowed to bud off into transport vesicles to be secreted.

Defects in protein folding are linked to diseases. For example, the most common mutation in Cystic Fibrosis ( $CFTR\Delta 508$ ) involves a protein stuck in the ER, affecting the Conductance Channel.
Misfolded proteins stimulate the Unfolded Protein Response (UPR), which constitutes a stress response:
- It upregulates ER chaperones to improve folding capacity.
- It triggers increased protein degradation.
- It causes a generalized stop in protein synthesis to prevent further accumulation of misfolded proteins.
If the UPR signal persists, it triggers apoptosis (cell death).
Cancer cells often hijack the UPR for survival, leading to chemoresistance. They may upregulate drug efflux pumps to remove chemotherapy chemicals from the cell.

Mitochondrial Matrix Import:
- Proteins utilize an N-terminal signal sequence recognized by the TOM complex in the outer membrane.
- The protein translocates through both the TOM complex and the TIM23 complex to enter the matrix, where the signal is cleaved.
- The signal sequence typically forms an amphipathic alpha helix. The receptor recognizes the hydrophobic face and structure of the helix rather than the specific amino acids.
Bacterial Outer Membrane:
- Translocation is similar to mitochondrial import. Involves the SAM complex for insertion into the mitochondrial outer membrane and the analogous BAM complex for insertion into the bacterial outer membrane, often forming beta-barrel structures.
Chloroplast Translocation:
- Uses the TOC complex (outer membrane) and TIC complex (inner membrane).
- Translocation into the stroma is GTP- or ATP-dependent.
- Getting into the thylakoid space requires an additional membrane crossing. There are four routes to the thylakoid space:
  1. Sec pathway (requires ATP and an H+ electrochemical gradient).
  2. SRP-like pathway (requires ATP and an H+ electrochemical gradient).
  3. TAT pathway (requires an H+ electrochemical gradient).
  4. Spontaneous insertion (no energy requirements listed).