Lecture 1: The Nucleus Flashcards
Learning objectives
- What are organelles and what do they enable a Eukaryotic cell to do?
- What are the fundamental principles and 3 methods of protein sorting?
- What is the structure and what are the functions of the nucleus?
- How does gated transport work?
Give experimental techniques for observing proteins.
- Proteins can be tracked using coloured beads
- Active cells can be chilled so that their protein sorting slows down and is easier to observe
How can proteins be moved around the cell?
- channels in membranes
- cytoskeleton, carrier or motor proteins
- vesicles
- energy source: GTP
- signalling sequence on protein and specific receptors at destination (if signalling sequence or receptors are mutated, protein does not go to correct place in cell, this can cause disease)
What are the three methods of protein sorting?
1) Gated transport (between the cytosol and nucleus)
2) Transmembrane transport (cytosol across membrane into a different space)
3) Vesicular transport ( vesicles between compartments)
What does the nucleus do?
- protects the genome
- replicates DNA
- transcribes DNA to form RNA
- regulates gene expression to control the rest of cell
How does the nucleus allow more complex genomes in eukaryotes than prokaryotes?
- The genome is protected
- More efficient DNA and RNA synthesis (and separate protein synthesis)
- more RNA processing is possible away from ribosomes, e.g. gene splicing
- Enables more complex gene regulation (gene splicing)
What is the structure of the nucleus?
- The nucleus has two membranes and many pores.
- The outer membrane has ribosomes and is continuous with the ER (perinuclear space is continuous with the ER)
- The nucleolus is an aggregate of macromolecules for ribosome synthesis (rRNA genes, rRNAs, enzymes, partly assembled ribosomes). The assembled ribosomes are then transported to the cytoplasm. The number and size of nucleoli varies with amount of protein synthesis and stage in cell cycle
- The nucleoplasm: gel-like ‘cytoplasm’ of nucleus (contains nucleotides, triphosphates, enzymes, transcription factors)
- Nuclear matrix - like cytoskeleton of cell
- Chromatin: DNA + proteins
- Nuclear speckles: helpful during extra active transcription, often found near heavily transcribed genes
- Nuclear lamina: scaffold or intermediate filaments called lamins, which are attached to the nuclear pores and integral membrane proteins, providing strength and stability for the nuclear envelope. If the lamins are mutated, the nucleus can crumple, which represses transcription and interferes with transport in and out of nucleus and causes the disease Progeria - the body becomes prematurely elderly and frail.
Describe nuclear pore complexes.
- Gated transport involves transport through the nuclear pore complexes (NPCs).
- It is carefully regulated to protect the genome and prevent harmful molecules entering the nucleus and damaging the DNA inside.
- The number of NPCs on the nucleus depends on the type of cell and stage in the life cycle.
- NPCs are made up of 30 nucleoporins (proteins).
- Molecules smaller then 5000 D called diffuse freely through the membrane, whilst molecules larger than 60,000 D is kept out by the disordered mesh.
- The nuclear pore complex has cytosolic fibrils and a nuclear basket region, with a disordered mesh in the centre, which is rich in hydrophobic amino acid residues.
- Proteins bounce through the disordered mesh into the nuclear basket.
- The nuclear pores are aqueous so can transport folded proteins (environment doesn’t change)
What is imported/exported into/from the nucleus?
Imported:
- Proteins (DNA polymerase, lamins)
- Carbohydrates
- Signalling molecules
- Lipids
Exported:
- RNA
- ribosomal proteins
What is the NLS?
The Nuclear Localisation Signal (NLS), which often has sequences which are positively charged (Lysine and Arginine). The NLS can be anywhere in the protein and is not cleaved off (maybe so protein returns to nucleus after cell division when nuclear envelope disintegrates). It can be masked (revealed only when another signal is received by the protein). NLS can be predicted from amino acid sequences.
Describe nuclear import.
Nuclear import is carried out by nuclear import receptors (alpha and beta importin). These are soluble and cytosolic. They bind to the NLS in the protein to be imported (sometimes an adaptor is also bound) and then bind to the FG (hydrophobic) repeats in the cytosolic fibrils of the nuclear pore. The importin-protein(-adaptor) complex moves through the disordered mesh in the pore and enters the nucleus, where the complex binds to Ran-GEF, which exchanges the GDP for GTP, causing the importin (+ adaptor) to dissociate from the protein. The importin moves back through the pore into the cytosol, where its Ran-GTP is converted back to Ran-GDP by Ran-GAP, making the importin ready to accept and transport another protein into the nucleus.
Nuclear import needs a power source: here it is GTP. A GTPase called Ran catalyses the hydrolysis from GTP to GDP and vice versa.
Ran-GAP is in the cytosol and converts Ran-GTP to Ran-GDP.
Ran-GEF (attached to the chromatin) is in the nucleus and converts Ran-GDP to Ran-GTP.
What is an NES?
A Nuclear Export Signal. It is a short amino acid sequence of 4 hydrophobic amino acid residues that targets a protein for export from the nucleus.
Describe nuclear export.
Nuclear import in reverse.
Exportins instead of importins.
GDP causes dissociation of protein instead of GTP.
