L10 - Compartmentalisation of the cell and protein sorting - gated transport and transmembrane transport

what is the nuclear pore?

• genetic material goes into and out of the nucleus via the nuclear pore

where is the nuclear pore formed and what is it made of?

• the nuclear pore is formed at the junction of the inner and outer membranes of the nuclear envelope

• the nuclear pore complex consists of multiple copies of around 30 different nucleotides

• each complex is made of 8 subunits with a central plug

where are the nuclear pore proteins?

• concentrated around the edge of the nucleus

what happens in a dividing cell when the nuclear envelope breaks down?

• the nuclear pore proteins are distributed throughout the cytoplasm

what are the nuclear pore complexes?

• large protein structures that span the nuclear envelope

• they regulate the movement of molecules in and out of the nucleus

what are the two ways substances can be transported via nuclear pore complexes?

• diffusion

• active transport

explain diffusion as a way of transport of substances via the nuclear pore complex

• materials can diffuse into the nucleus

• molecules with molecular weight up to 5000 = freely diffusible

• molecules with molecular weight up to 60,000 = slower diffusion rate

• any molecules above 60,000 = cannot enter via diffusion

explain active transport as a way of transporting substances via the nuclear pore complex

• requires input of energy (ATP)

• under appropriate signals the pore opens (up to 26nm in diameter)

what is the signal needed for the active transport of proteins?

• signal linked to a peptide sequence

• like a sequence of amino acids or a structural arrangement within the protein

what is the signal needed for active transport into the nucleus?

• nuclear localisation signal

• rich in positively charged amino acids

• the nuclear pore has proteins that recognise sequences of amino acids rich in lysine, arginine, proline

what would happen if an amino acid was mutated in the sequence of T-antigen of the SV40 virus?

• the amino acids are important for transport of the SV40 into the nucleus

• if one of the amino acids is mutated then it prevents movement of the T-antigen (SV40) into the nucleus

• disrupt sequence = disruption of signal

what are two evidence to support that transport is active?

1. mRNA transport into the nucleus

   when the cells were cooled down to 4C the process was inhibited

2. in vitro

   in absence of ATP - the protein binds to nuclear pore but remains outside of nucleus

   in addition of ATP - the proteins started to appear inside the nucleus

Endoplasmic reticulum (ER)

• ribosomes found on ER

• the ribosomes are the site of synthesis for the membrane proteins within the cell

• the ribosomes are tightly opposed to the ER

what is ER translocation?

the process by which newly made proteins are moved (translocated) into or across the endoplasmic reticulum (ER) membrane

what are the two ways newly made proteins can be translocated into organelles?

1. co-translationally

2. post-translationally

what is co-translational translocation?

• when the protein is moved into the ER while it’s being synthesised by the ribosomes (being made)

• translation and translocation happen at the same time

what is post-translational translocation?

• when the protein is fully made first and then moved into the ER after translation is complete

• translation happens first before translocation

what is the signal hypothesis?

• states that translocation into the ER requires a signal

• meaning proteins must have a signal in order to be translocated

• proteins that are meant to go into the ER have a built in signal sequence which directs them as they are being made

what are the steps in co-translational translocation?

1. translation begins on a free ribosome in the cytosol

2. a signal peptide emerges directing the ribosome to the ER

3. ribosome binds to the ER via a translocator (Sec61)

4. the growing polypeptide is threaded into the ER lumen

5. signal peptidase (enzyme) cleaves off the signal sequence

6. the mature protein is then released into the ER lumen

7. the signal is also released and gets broken down

what is the translocator Sec61?

• a protein channel in the ER membrane that allows newly made proteins to enter the ER during co-translational translocation

what are the two different conformations that the Sec61 translocator exists in?

1. closed conformation

2. open conformation

what is the closed conformation of translocator Sec61?

• no ribosome is bound and no protein is being made

• channel (Sec61) is shut preventing unwanted molecules/ions from leaking through the ER membrane

what is the open conformation of translocator Sec61?

• triggered when a ribosome binds to Sec61

• a signal peptide is recognised

• channel opens

what is meant by ribosome bound but closed when referring to the Sec61 translocator?

• it means that the ribosome is docked but no translocation is happening yet because the pore is still shut until a signal peptide is recognised

how are membrane proteins inserted into the ER?

1. signal allows movement into ER

2. signal is recognised by translocation machinery

3. instead of transferring the whole polypeptide through there’s a stop transfer sequence

4. when this stop transfer sequence reaches the translocator it stops the transfer

5. signal peptidase comes along and removes the signal sequence and releases the membrane anchored protein into the ER membrane

what does the insertion of single pass transmembrane proteins depend on?

• these proteins span the membrane once

• their insertion depends on the type of signal sequence + orientation (topology)

type 1 membrane protein topology

• N terminal in ER lumen

• C terminal in cytosol

• has a cleavable N terminal signal peptide

• uses a strop transfer sequence to anchor in membrane

type 2 membrane protein topology

• N terminal in cytosol

• C terminal in ER lumen

• has an internal signal anchor sequence - acts as both signal + membrane anchor

what are chaperones?

• proteins in the ER which ensure proteins fold properly

what can defects in protein folding (misfolded proteins) lead to?

• disease

• example - CFTRdelta508 mutation in cystic fibrosis

• misfolded proteins stimulate the unfolded protein response

what is the unfolded protein response?

• when too many misfolded proteins accumulate in the ER this response helps to prevent damage to the cell

how are proteins transported into the mitochondrial matrix (post-translational translocation)?

• the protein is fed through 2 complexes - through one pore onto the next pore

• once translocation has occurred the signal peptidase cleaves off the signal

• mature mitochondrial protein in inner space

what is the structure of the signal in post-translational translocation?

• composed of amphipathic alpha helix with hydrophobic residues

• recognises hydrophobic grooves

process of protein translocation via post-translational translocation

1. protein is made in the cytosol with a signal sequence

2. chaperones keep the protein unfolded

3. signal sequence is recognised by a receptor on the target membrane

4. protein is translated through membrane complexes

5. chaperones inside help pull in and fold the protein

6. signal sequence is cleaved off

steps of post-translational translocation

1. protein is fully made first in the cytosol

2. kept unfolded by cytosolic chaperones

3. guided to translocator Sec61

4. pulled into ER after translation with help from ER chaperones

5. signal sequence cleaved off