Intracellular compartments and protein sorting

Question 1

What are the different ways through which proteins can be moved and sorted?

Answer 1

Gated transport
Protein translocation
Vesicular transport

Question 2

Describe the general process of vesicular transport.

Answer 2

A donor compartment with proteins inside where vesicles originate.

This vesicle has its own cargo and is made of the same type of membrane as the donor compartment, as well as being target marked by surface molecules present on the membrane of both the donor compartment and the vesicle.

This then reaches the target compartment, fuses with it and releases its cargo in the compartment.

Question 3

How is the transported protein sorted to the correct destination?

Answer 3

The transported protein contains a signal which is used by the signal to correctly sort the protein into its destination.

A stretch of hydrophobic amino acids are important for the import of proteins into the ER.

Also to be imported into the nucleus, a specific stretch of amino acids are needed, which are positively charged.

Question 4

What molecules are directed to the nucleus?

Answer 4

Nuclear localisation signals direct nuclear proteins to the nucleus.

All proteins produced in the cytoplasm will then perform their function in the nucleus. Together with this, RNA molecules are also produced and moved around in the nucleus.

Question 5

Describe the structure of the nuclear pore?

Answer 5

Nuclear pore can be easily observed. It has 2 different faces, the external and the internal facing sides.

They have a very complex function able to recognise different molecules and RNA extensions.

The outer part of the nuclear pore is constituted by the cytosolic fibrils exposed to the cytoplasm, while the inner part facing the nuclear plasma has a basket shape.

The pores are large, expandable and aqueous, so the proteins dont need to lose their shape when they pass through.

Question 6

What complexes are assembled in the nucleus?

Answer 6

Large complexes such as ribosomal subunits are assembled in the nucleus, then go back to the cytoplasm.

In this case, they will have another type of signal, an export signal.

Question 7

What happens if the nuclear importing signal is mutated?

Answer 7

If a nuclear importing signal is mutated, the protein is unable to enter the nucleus and thus stays in the cytoplasm.

Question 8

Describe the receptors needed for nuclear import and export.

Answer 8

A receptor is also needed to receive the signal for nuclear import. This general class is called importins, encoded by a family of related genes. Each of them can import a subset of proteins.

In some cases receptors can bind directly to the cargo and an intermediate is needed.

Exportin receptors are needed to receive the signal for nuclear export. Exportins and importins are very similar both in structure and sequence.

Question 9

How is energy transformed for nuclear export and import?

Answer 9

Energy is needed, as well as other proteins. Ran proteins shuttle between cytosol and nucleus, changing their state. When they are in the cytosol, ran proteins are bound to GDP, while inside the nucleus, they are bound to GDP.

GAP is a soluble protein present in the cytosol. It changes the state from GTP to Ran-GDP.

While GEF is not soluble and stably bound to chromatin in the nucleus, it changes the state from GDP to GTP.

Question 10

Describe the process of nuclear export.

Answer 10

A protein with its specific nuclear export signal bound to a nuclear export receptor, this can only take place in the presence of ran GTP.

This complex passes through the nuclear pore along the cytosolic fibrils into the cytoplasm. This complex is dissociated, the protein is free to travel in the cytoplasm while the exportin is released and recycled as ran GTP becomes ran GDP.

Question 11

Describe the process of nuclear import.

Answer 11

A target protein will have a specific nuclear localisation signal, which is recognized by a nuclear import receptor, This happens in the near proximity with cytosolic fibrils present in the nuclear pore. Only by binding to this is the complex driven through the nuclear pore.

Once entered into the nucleus, this complex meets Ran GTP. This displaced the cargo protein from the importin receptor. Cargo protein is delivered and ran GTP binds with importin to go back to cytoplasm to be used again.

Ran GTP in the cytoplasm is turned into ran GDP + organisa P.

Question 12

Describe generally how mitochondrial proteins translocates into the mitochondria.

Answer 12

Mitochondrial proteins are synthesized in the cytosol as precursor proteins, then translocated to the mitochondria in a translocational mechanism.

To do this, a signal sequence is needed. These are typically located at the N terminal of the mitochondrial proteins. Once used, they will be removed by a signal peptidase.

Although other proteins, including outer membrane, inner membrane and intermembrane space proteins, have internal signal sequences that are not removed.

Question 13

Describe the structure of the signal sequences that direct proteins into the mitochondrial matrix.

Answer 13

The signal sequence forms an amphiphilic alpha helix, with + residues cluster on one side of the helix, while uncharged hydrophobic residues on the opposite side.

Question 14

Describe the process of the mitochondrial protein translocation into the mitochondria.

Answer 14

Proteins from the cytoplasm has a specific signal sequence in the N terminal, which is recognized by the TOM receptor.

This drives its passage through the large TOM complex. Then the protein slides through the TIM23 complex to enter the matrix.

In the matrix, its signal sequence is cleaved by a signal peptidase.

Question 15

Describe the complexes in the mitochondrial membrane involved in protein translocation.

Answer 15

Outer mitochondria membrane:

TOM complex transfers proteins across the outer membrane.

SAM complex helps proteins to fold properly in the outer membrane. This is for proteins that remain in the outer membrane.

Inner mitochondrial membrane:

TIM 22 complex mediates the insertion of a sub-class of inner membrane protein, which moves ATP, ADP and phosphate in and out of the mitochondrial membrane.

TIM 23 complex transports soluble proteins into the matrix and helps insert transmembrane proteins into the inner membrane.

OXA complex mediates the insertion of inner membrane proteins synthesized in the mitochondria.

Question 16

Describe the main features of peroxisomes.

Answer 16

Contains oxidative enzymes so concentrated that peroxisomes can be observed under TEM in some cells.

Peroxisomes use molecular oxygen and hydrogen peroxide to perform oxidation reactions. Catalase uses H2O2 to oxidase other substrates, e.g. alcohol.

Especially liver and kidney as used to detoxify molecules.

Concentration of H2O2 needs to be regulated, so when excess H2O2 occurs, catalase converts it to oxygen.

Question 17

Describe the functions of peroxisomes.

Answer 17

A major physiological function is the breakdown of specific fatty acid molecules (beta oxidation).

Chains of fatty acids are cut to make acetyl coA which is recycled for use in biosynthetic reactions. In mammalian cells, beta oxidation also occurs in the mitochondria.

Another important function is synthesis of plasmalogens. These are phospholipid, important in the composition of myelin which is important for the transmission of nervous signals.

Deficiency in synthesis of this causes diseases. Therefore many types of disorders of peroxisomal dysfunctions end up in neurological diseases.

Question 18

Describe Zellweger syndrome.

Answer 18

An inherited human disease where there is a mutation in one gene which encodes for peroxin. Consequently the import of proteins into peroxisomes are dysfunctional.

This results in ‘empty peroxisomes’ and severe abnormalities in the organs where peroxisome function is essential, for instance brain, liver and kidneys.

This leads to death soon after birth.

Question 19

Describe the process of import into the peroxisome.

Answer 19

The import signal is a specific sequence of 3 amino acids located at the C terminus of many peroxisomal proteins.

Peroxins use ATP hydrolysis to drive the import process. They form a complex of at least 6 different peroxins resulting in a protein translocator in the peroxisome membrane.

This pore is dynamic in dimension and adapts to the cargo, therefore proteins don’t need to unfold.

Question 20

Describe the ER.

Answer 20

All eukaryotic cells have ER, its size is large in animals.

Labyrinth of tubules and sacs that extend throughout the whole cytosol.

The ER and nuclear membrane forms a continious sheet enclosing a single internal space, called the ER lumen (ER cisternal space). This occupies more than 10% of total cell volume.

Question 21

What kind of proteins need to enter the ER?

Answer 21

All proteins that enter the ER are synthesised in the ribosomes, where they are directed to the ER via an ER signalling sequence.

Transmembrane proteins: partly translocates across the ER membrane and becomes embedded.

Water-soluble proteins: fully translocates across the ER membrane and released in the lumen. Then its either secreted or remains in the ER.

Question 22

Describe the signal hypothesis.

Answer 22

During the translation of mRNAs by ribosomes, the forming polypeptide exposes the N terminal part containing its signal sequence.

The is recognised by a translocator receptor. Translocation starts whilst the polypeptide chain is being formed.

The signal sequence remains bound to the polypeptide as the chain is forming and sliding through the translocators.

Once it enters into the lumen, a signal peptidase cleaves the signal peptide, which remains in the membrane before being sent to degrade.

Question 23

How is the ER signal sequence guided to the ER membrane.

Answer 23

A signal-recognition particle (SRP) directs the ER signal sequence to a specific receptor in the RER.

The SRP cycles between the cytosol and binds to the signal sequence, and an SRP receptor in the ER membrane.

Question 24

Describe the structure of SRP receptor.

Answer 24

The SRP is a large complex.

In animal cells, it has 6 different polypeptide chains bound to a single small RNA.

Question 25

Describe the ER signal sequence.

Answer 25

Each has 8 or more non-polar amino acids at its centre.

Question 26

How does SRP work to guide the ER signal sequence to the ER membrane?

Answer 26

One end of SRP binds to the signal sequence just during its formation when it emerges from the ribosome.

The other side of the SRP blocks the binding site at the interface between large and small ribosomal subunit, resulting in a transient pause.

Presumably this is to give ribosome time to bind to to the ER membrane before the polypeptide chain is completed, to avoid it being released into the cytosol.

Question 27

After the SRP has guided the ER signal sequence to the ER membrane, how does the polypeptide pass into the lumen?

Answer 27

A protein translocator is also needed and the channel through the polypeptide chain will pass through the channel.

The signal peptide exposed from the ribosome binds to a site inside the translocator and remains there whilst the rest of the protein (still being produced) slides through the translocator and enters into the lumen.

After the signal peptide has bound to the translocator, the SRP is released.

Question 28

Describe single-pass transmembrane proteins.

Answer 28

An N terminal signal sequence function as a start-transfer signal, to start the translocation process by opening the translocator.

The sequence shifts laterally into the lipid bilayer before another signal, called stop-transfer sequence, binds with a specific site in the translocator to stop the passage of the protein.

Consequently the protein is anchored to the membrane.

Question 29

Describe multi-pass transmembrane proteins.

Answer 29

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Question 30

What is a retention signal?

Answer 30

Needed for the protein to remain in the ER.

This is usually a short sequence of amino acids located at the C terminal of the polypeptide chain.

Question 31

Describe the fate of proteins in the ER.

Answer 31

Many proteins are in transit to other membrane bound organelles.

While some are retained. The role of many of these retained proteins are catalysts that work in keeping unfolded proteins in the lumen of the ER until they are correctly folded.

Question 32

Describe how proteins are processed in the ER.

Answer 32

Abount half of soluble and membrane bound proteins processed in the ER are glycoproteins.

Most proteins are glycosylated by an addition of N-linked oligosaccharide.

Question 33

How are oligosaccharides used as tags to mark the state of protein folding (in the ER)?

Answer 33

In multipass transmembrane proteins, it’s based on the presence of more than one start and stop transfer sequences.

the start-transfer sequence could also not be at the end, but in the middle of the polypeptide chain.

Question 34

What happens in the ER when there is a large amount of unfolded proteins?

Answer 34

If there is a large amount of unfolded proteins, a response is activated called unfolded proteins response.

This results in an increased gene transcription of genes encoding for proteins that help to increase the folding capacity of the ER.

Question 35

What happens if folding isn’t possible in the ER?

Answer 35

If folding isn’t possible, the proteins are transported back into the cytosol and degraded by proteasomes.

These failures are very common. Amongst the large amount of proteins the cell produces, only a few works. Therefore this process is continuously working.

Question 36

How does protein folding in the ER play a part in cystic fibrosis?

Answer 36

Cystic fibrosis is an inherited disease caused by mutations of a membrane protein working as a chloride channel.

Cystic fibrosis happens when this protein doesn’t work.

This disease is recognized as mutant by the ER, therefore it does not allow the mutated protein to reach its final location and is degraded. Although the mutant protein, if allowed to reach the membrane, would still work.

Question 37

Describe the functions of the ER that are related to lipids.

Answer 37

Specific ER enzymes covalently attach a GPI anchor to the C terminus of some membrane proteins designated for the PM.

This linkage forms in the lumen where the transmembrane segment of the protein is cleaves off at the same time.

Many PM proteins are attached this way. Therefore they can be released from cells in response to signals that activate a specific phospholipase in the PM.

Question 38

What are trypanasomes?

Answer 38

A parasite that sheds their coat of GPI anchored surface proteins when attacked by the immune system.

Question 39

Describe how the ER can synthesise lipids.

Answer 39

The ER synthesises nearly all of the cell’s major classes of proteins. One of the major phospholipids is phosphatidylcholine.

The synthesis happens in the membrane.

Fatty acids arrive from the cytosol, it needs to be activated by bonding to CoA.

Acyl transferase adds 2 fatty acids to glycerol phosphate to produce phosphatidic acid.

Phosphate is released leaving diacylglycerol.

Choline binds to the 3rd C group via choline transferase.