Lecture 9 - compartmentalisation of the cell and protein sorting Flashcards
What is the advantage of compartmentalisation?
compartmentalisation increases the number of functions that a cell can perform
- each feature has its own protein/lipid composition
- proteins hade in the cytosol
- use transport to travel to areas they are required - gated and transmembrane transport
- in order for a protein to reach its intended target, it requires a signal
Describe features of the nuclear pore
Double membrane structure - inner & outer membrane. This acts as a barrier to keep DNA inside nuclear.
- formed at the junction of the inner and outer membranes of nuclear envelope
- nuclear pore complex is not a simple hole - the complex consists of multiple copies of approx 30 different nucleoporins
- each complex appears to be made of 8 subunits with a central plug
What has cryoelectronic microscopy allowed?
allowed a model of how the pore is organized in high structural detail. It can change size to allow different sized cargo into the nucleus
What do the nuclear pore complexes do?
Involved in moving substances across the nuclear envelope
Examples:
- in the synthesis of DNA, histone molecules are needed to package the new DNA. These are transported from the cytoplasm.
For protein production to occur, ribosomes are needed. The ribosomal subunits formed in the nucleolus have to enter the cytoplasm.
- the pore is variable size, so diffusion can occur
What 2 ways can substances be transported by the pore complex?
- diffusion
- active transport
Until what weight can molecules cross the pore complex?
60,000
How wide can a pore open for active transport to occur under appropriate signalling?
26nm in diameter
What signals lead to gated nuclear import?
In the case of proteins - signal is linked to a peptide sequence. Nuclear transport recognition sites rich in Lys, Arg and Pro (Lysine, Arginine and Proline)
Example - T-antigen of the SV40 virus contains sequence - Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val. This sequence is important for transport of the antigen into the nucleus
How can we prove that a signal allows transport?
- Sequence intact - localised in the nucleus
2, Sequence disrupted - no staining in the nucleus
What is experimental evidence supporting active transport in cells?
mRNA transport into the nucleus - inhibited on cooling to 4 degrees Celsuis
How is ATP hydrolysis required in vitro?
in the absence of ATP, the protein binds to the pore complex but remains outside the nucleus. If you add ATP, the proteins start to appear inside the nucleus
Describe ER translocation
Ribosomes are site of synthesis for membrane proteins inside the cell. Very close to the ER membrane
When are newly made protein translocated into organelles?
Co- or post-translationally
How does translocation into the ER require a signal?
Ribosomes translate mRNA, sitting close on the ER membrane. Signal allows interaction with translocator. Linear sequence of amino acids are fed into lumen of ER. Signal Peptidase (like molecular scissors) snips off signal). The signal released from newly translated protein, and the protein is then released into the ER lumen and the signal is released from the translocator and get broken down.
How are membrane proteins inserted into the ER?
Signal is recognised by translocation machinery, but instead of feeding whole polypeptide through, there is a stop transfer sequence, which when reaches the translocator, it stops transfer and the signal peptidase removes the signal sequence and releases the membrane protein into the ER membrane.
