Intracellular Transport Lecture Notes

Intracellular Transport

• Intracellular Transport 1 (today)
• Intracellular Transport 2 (Tuesday)
• Cytoskeleton
• Cell Cycle
• Cancer Lecture
• Review
• Final (non-cumulative)

Overview

• Focus: How things move around in the cell, especially in and out of organelles.
• Three organelles of primary focus: Endoplasmic reticulum (ER), Golgi apparatus, and plasma membrane.

Protein Sorting

• Today's lecture could be reframed as protein sorting, namely how proteins are directed to specific locations.
• There will be three strategies discussed today and in the next lecture by which proteins are distributed into different organelles.

Consequences of Mislocalization

• Mislocalization of proteins can lead to severe diseases, including developmental problems and potentially deadly conditions.

Three different strategies

• Today: Learn two strategies for moving things around inside the cell.
• Next lecture: Strategy three, involving vesicles.

Membranes and Transport

• Eukaryotic cells contain organelles that are enclosed by membranes.
• These membranes provide protection and control the movement of substances in and out.
• Movement is mediated by import and export mechanisms.

Protein Sorting Definition

• Proteins are distributed to specific organelles or between organelles.
• This process may involve import, export, or both.
• Proteins often move through multiple organelles.
• Protein sorting requires a special sequence within the protein known as a signal sequence.
• The signal sequence is essential for directing the protein to its correct destination.
• Signal sequences can range from 3 to 60 amino acids in length.

Vesicles

• Vesicles are lipid-derived membrane structures that act as cargo carriers, transporting proteins from one organelle to another.
• Vesicles are made of phospholipids.
• Vesicular transport: Using vesicles to transport things from one place to another
• Motor proteins (kinesin and dynein) carry vesicles through vesicular transport from one organelle to the other.

Vesicular Transport

• ER to Golgi: Vesicles are heavily utilized.
• Golgi to Plasma Membrane: Vesicles are also a major form of transport.

Protein Sorting Requirements

• Proteins must have signal sequences to be imported or exported.
• Signal sequences vary, ranging from approximately 3 to 60 amino acids.

Translation and Protein Sorting

• Translation: Proteins are made, assessed, and then directed to specific locations.
• Protein sorting determines where proteins are sent and how they are transported.
• Old or damaged proteins are replaced through continuous import and export processes.

Transport Mechanisms

• Translocators: Channels or tunnels that allow select proteins to pass through, with a specific sequence of events determining which proteins can cross.
• Pores: Organized holes that facilitate movement.
• Vesicles: Lipid-derived membranes for transport
• Organelles may favor using pores, translocators, or vesicles.
• ER to Golgi and Golgi to plasma membrane extensively use vesicles.

Key Strategies

• Pores: Essential for the nucleus.
• Translocators: Essential for the mitochondria, chloroplasts, and ER.
• Vesicles: Discussed in the next lecture.
• Whether using a pore, translocator, or vesicle, a protein must have a signal sequence for entry.

Nuclear Pore Complex (NPC)

• Pores act as barriers between the cytosol and the nucleus.
• Proteins and other molecules (e.g., mRNA) must pass through these pores to enter or exit the nucleus.
• mRNA uses these pores to leave the nucleus.

Signal Sequences

• Different signal sequences have different functions and specific amino acid chains.
• Nuclear Import Sequence: Directs proteins into the nucleus.
• Mitochondrial Import Sequence: Directs proteins into the mitochondria.
• Mutations in signal sequences can prevent proteins from reaching their destinations, leading to cellular dysfunction and potentially apoptosis.

Nuclear Transport

• Proteins entering the nucleus must pass through pores.
• Nuclear Localization Signal (NLS): A specific signal sequence required for nuclear import.
• Proteins with an NLS undergo cargo shuttling.
• The nuclear envelope surrounds the nucleus and is supported by intermediate filaments.

Nuclear Pore Complex (NPC) Structure

• The pore is formed by proteins called nucleoporins.
• Nucleoporins make up the nuclear pore complex (NPC).
• Cytosolic fibrils act as tethering sites.
• Nuclear Import Receptor (NIR): Located on the outside of the nucleus, recognizes and binds to the NLS on proteins targeted for import.

Nuclear Import Mechanism

• NIR binds to the NLS of the protein intended for import.
• The NIR-protein complex is tethered to cytosolic fibrils.
• The complex is then transported across the nuclear pore.
• Once inside the nucleus, the NIR dissociates from the protein of interest.

Key Molecules in Nuclear Transport

• Ran-GDP: An essential shuttling molecule that helps the NIR-protein complex cross the nuclear pore.
• Ran-GEF: Located inside the nucleus, converts Ran-GDP to Ran-GTP, causing the NIR and protein to dissociate.
• Ran-GAP: Located outside the nucleus, promotes the hydrolysis of Ran-GTP to Ran-GDP.

Nuclear Import Cycle

1. NIR (Nuclear Import Receptor) recognizes the NLS (Nuclear Localization Signal) on the target protein.
2. The complex of NIR and the target protein comes across with help from Ran-GDP (RAndom nucleotide-binding protein - Guanosine DiPhosphate).
3. Inside the nucleus, Ran-GEF (Guanine nucleotide Exchange Factor) swaps GDP for GTP (Guanosine TriPhosphate) on Ran, converting it to Ran-GTP.
4. This conversion causes the NIR to release the target protein.
5. The target protein remains in the nucleus while the NIR and Ran-GTP leave through the nuclear pore.
6. Outside the nucleus, Ran-GAP (GTPase Activating Protein) stimulates hydrolysis of GTP to GDP on Ran, converting it back to Ran-GDP.
7. The NIR and Ran-GDP are now ready to repeat the cycle by binding another target protein with an NLS.

Mitochondrial Protein Import

• Proteins targeted for the mitochondria have a mitochondrial import sequence.
• Mitochondrial Import Receptor: Recognizes the mitochondrial import sequence.
• Translocators: Channels through which proteins cross the mitochondrial membranes.
• The protein is unfolded before crossing the translocator.
• Mitochondria have two membranes: an outer and an inner membrane.

Steps of Mitochondrial Import

1. The mitochondrial import receptor binds to the mitochondrial import sequence on the protein.
2. The protein is unfolded and translocated across the outer membrane via translocator one.
3. The protein is translocated across the inner membrane via translocator two.
4. The protein enters the mitochondrial matrix.
5. Chaperone proteins help refold the protein.
6. Signal peptidase cleaves off the import sequence.

Endoplasmic Reticulum (ER) Protein Import

• Proteins are made on ribosomes that are attached to the rough ER.
• As the protein is made, it immediately enters the ER through an import mechanism.
• ER lumen: The space inside the ER.

ER Import Mechanism

• The new polypeptide (protein) is about to enter the ER.
• ER Signal Sequence: A sequence on the protein that signals it to be imported into the ER.
• Signal Recognition Particle (SRP): Recognizes and binds to the ER signal sequence.
• SRP Receptor: Binds to the SRP, bringing the protein to the ER membrane.

Steps of ER Import

1. As the polypeptide is synthesized on the ribosome, the SRP binds to the ER signal sequence.
2. The SRP brings the ribosome to the SRP receptor on the ER membrane.
3. The protein begins to be translocated across the ER membrane through a translocator.
4. Once inside the ER lumen, the ER signal sequence is cleaved off by signal peptidase.
5. The protein can then either stay in the ER, move to the Golgi, or go beyond to the plasma membrane.

Start-Stop Transfer Sequence

• If a protein is destined to be in the ER membrane, it will have a start-stop transfer sequence.
• The protein begins to be translocated across the ER membrane, but then the transfer is stopped.
• The signal peptidase cleaves off the start sequence, revealing a second sequence that causes the protein to stop in the ER membrane.
• This mechanism allows the protein to be efficiently inserted into the ER membrane without being fully translocated into the ER lumen.