Intracellular Protein trafficking

Question 1

Where do majority of proteins start their journey to their target locations?

Answer 1

Synthesised in the nucleus –> Cytosol –> target destinations

Question 2

What are sorting signals? What are the 2 types of sorting signals?

Answer 2

Sorting signals - protein regions that determine its destination in the cell

1) Signal sequence - exposed single stretch of AAs, often at the end of the polypeptide chain
2) Signal patch - a region of a protein only recognisable after protein folding

Question 3

What is the nuclear import sorting signal?

Answer 3

lysine & arginine-rich sequence

- can be a signal or a patch

Question 4

How do proteins of different sizes access the nucleus?

Answer 4

o Small molecules (< 5 kdaltons) can diffuse freely through nuclear pores
o Larger molecules are imported via active transport

Question 5

What are nucleoporins?

Answer 5

Proteins making up the nuclear pore complexes

Question 6

Describe the process of nuclear protein import.

Answer 6

The protein NLS binds to the Importin (free-floating cytosolic receptor). Importin guides the protein through the channel by binding to FG repeats on nucleoporins.

Question 7

Describe the process of nuclear protein export.

Answer 7

Exportin (free floating nuclear receptor) binds to the NLS of the protein. Exportin guides the protein through the channel by binding to exportin binding sites on nucleoporins.

Question 8

What is the role of Ran-GTPase in nuclear protein transport?

Answer 8

Ran-GTPase establishes the gradient of GTP across the nuclear membrane through the action of 2 proteins.
o Cytosolic Ran-GAP - a GTPase-activating protein, catalysing the conversion of cytosolically-bound RanGTP to RanGDP
o Nuclear Ran-GEF -
a protein that converts RanGDP to RanGTP

Question 9

Describe how Ran-GTPase mediates the protein import across the nuclear membrane.

Answer 9

In the cytosol Importin dissociates with Ran-GDP and Pi in and binds the cargo protein, which allows for its transport across the membrane. In the nucleus the cargo protein is released through a Ran-GTP binding to the importin, and the Ran-GTP-associated Importin travels back to the cytosol through the nuclear membrane.

Question 10

Describe how Ran-GTPase mediates the protein export across the nuclear membrane.

Answer 10

In the nucleus Ran-GTP binds to the exportin, which allows for binding of the cargo protein. That complex travels across the nuclear membrane to the cytosol, where Ran-GDP and Pi dissociate and the cargo protein is released.

Question 11

What is the mitochondrial import sorting signal?

Answer 11

amphipathic alpha helix

Question 12

What is mitochondrial translocation and where does it occur?

Answer 12

It is the transport of proteins to the mitochondria at points where the inner and outer mitochondrial membranes are the closest to each other

Question 13

What is the protein-confirmation requirement for mitochondrial translocation? What protein is involved in achieving this requirement?

Answer 13

Proteins need to be kept unfolded after translation. Cytosolic Hsp70 keeps the proteins unfolded with the use of ATP.

Question 14

Describe how proteins are translocated to the mitochondrial matrix.

Answer 14

Signal sequence binds to the membrane-bound receptor associated with TOM(translocon of the outer membrane) complex. The unfolded polypeptide is translocated through TOM into the space between the membranes, where it is guided towards the TIM(translocon of the inner membrane) complex. Transport through the TIM requires an electrochemical gradient for transportation of proteins across the inner mitochondrial membrane.

Question 15

How are proteins edited in the mitochondrial matrix?

Answer 15

The signal sequence is cleaved by a protease and Hsp60 helps the translocated protein to fold properly in the matrix.

Question 16

How are proteins inserted into the outer mitochondrial membrane?

Answer 16

An unfolded protein goes through the TOM complex to the intermembrane space, where it is folded by chaperones and inserted into the outer mitochondrial membrane by SAM complex.

Question 17

What are the 3 pathways, through which proteins can be targeted to the inner mitochondrial membrane?

Answer 17

1) A protein goes through the TOM complex and its signal goes through the TIM complex as well. In the mitochondrial matrix, the signal sequence is cleaved and the following hydrophobic sequence stays embedded in the inner mitochondrial membrane while the rest of protein stay in the intermembrane space.
2) A protein goes through the TOM and TIM complexes, signal sequence is cleaved and the following hydrophobic sequence binds to the OXA complex, which allows the rest of the protein to be transported back to the intermembrane space, while the hydrophobic part anchors it to the inner membrane.
3) A protein goes through the TOM complex, where chaperones fold it and TIM22 complex insert it to the inner mitochondrial membrane.

Question 18

How can a protein be targeted to the intermembrane space?

Answer 18

Any protein anchored in the inner mitochondrial membrane can be cleaved off its hydrophobic domain by a protease, thus releasing it to the intermembrane space.

Question 19

What is the ER import sorting signal?

Answer 19

1 or more positively charged AAs followed by a hydrophobic AA sequence

Question 20

When and where does the translation occur in the transport to the ER? How this type of import is called?

Answer 20

Translation can either occur before the translocation in the cytoplasm (the protein needs to be kept unfolded) or the protein can be translated by a ribosome located right next to the ER membrane and translocated during translation. This type of translocation is called Cotranslational import.

Question 21

Describe the process of cotranslational translocation of a protein into the ER.

Answer 21

An N-terminus of a translated protein binds to the free-floating SRP receptor and the translation stops. SRP-bound ribosome attaches to the SRP receptor on the ER membrane. GTP hydrolisis causes the binding of the nascent protein end to the translocon complex and release of the SRP. The translation is continued and the synthesised protein is pushed through the Sec61 channel complex.

Question 22

What is the difference in the energy source requirement for cotranlational and posttranslational import.

Answer 22

cotranslational - requires GTP

posttranslational - requires ATP

Question 23

What is the mechanisms driving the posttranslational protein import into the ER?

Answer 23

The BIP proteins, with the use of ATP bind to the parts of the protein in the ER matrix stopping it from sliding back.

Question 24

Describe how a soluble protein is translocated into the ER lumen.

Answer 24

The protein sorting signal(hydrophobic) binds and activates the receptor, which upon activation starts pushing the unbound part of the protein inside the ER. Receptor-associated signal peptidase cleaves the signal sequence, which stays in the membrane, while the rest of the protein stays in the ER lumen.

Question 25

Describe the translocation of a membrane protein into the ER membrane.

Answer 25

The protein sorting signal binds and activates the receptor, which upon activation starts pushing the unbound part of the protein inside the ER, until it reaches a hydrophobic STOP region. Receptor-associated signal peptidase cleaves the signal sequence, leaving the STOP sequence in the membrane and soluble parts of protein on both parts of the membrane.

Question 26

What is N-linked glycosylation and where does it occur?

Answer 26

It is the addition of sugar to the protein on the Aspargine residues on sequences NXT or NXS (t- theronine, s-serine) in the ER.

Question 27

What is the other important protein modification occuring in the ER?

Answer 27

Formation of disulphate bonds between cysteine residues in the oxidative environment.

Question 28

What is ERAD?

Answer 28

ER-associated degradation of misfolded proteins which are transported back out of the ER and degraded in the cytosol.

Question 29

What are the 3 methods to study the protein translocation across the membranes and what are they used for?

Answer 29

1) Transfection approach - used to identify signal sequences - comparison of the distribution of GFP labelled protein and its modified version across the cell.
2) Biochemical approach - uses copurification to identify if the proteins are non-covalently complexed with certain organelles.
3) Genetic approach - uses yeast models to examine the eukaryotic protein pathways.

Question 30

How does direct fusion protein transport work? Example?

Answer 30

Proteins are transported from one organelle to another by fusion of their membranes and mixing of their intraornanellar contents. e.g. late endosome/lysosome fusion

Question 31

In vesicular transport the clathrin-coated vesicles traffic between which compatments?

Answer 31

Golgi -> endosome, lysosome, melanosome and platelet vesicles
Plasma membrane -> endosome

Question 32

What are vesicular coats made of and what is their function?

Answer 32

They are made of proteins embedded in the vesicular membrane. Their function is to assist vesicular formation (structural) and select cargo proteins (selective)

Question 33

What recruits the vesicular coats?

Answer 33

Specific GTPases recruit specific coats.

• Sar1 recruits COPII
• ARF recruits COPI & Cathrin-coated vesicles

Question 34

Describe the process of formation of the clathrin-coated vesicle.

Answer 34

Cargo molecule binds to the receptor -> bound receptor is recognised by the adaptor proteins -> adaptors proteins recruit clathrins -> adaptors and clathrin form a cage like structure -> the neck is pinched off by a GTPase -> ready vesicle -> as soon as it fully forms, the vesicle uncoats with a ATPase -> naked target vesicle, which will fuse with its

Question 35

What mechanism mediates the fusion of the vesicle with its specific target? What other fusion does it also mediate?

Answer 35

The fusion of vesicle with its target is mediated by the binding of a particular set of vSNAREs specific for a particular set of tSNAREs. This mechanism also mediates the organelle fusion.

Question 36

Describe the mechanism of vesicle-target fusion.

Answer 36

1) Tethering the vesicle to the target membrane
a. Requires tethering factors – Rab factors (small GTPases)
b. Rab GTP (specific for different membrane fusions) binds to its effector tethering the vesicle
2) The v-SNARE (α-helical structure) interacts with t-SNAREs forming a 4-α-helical bundle bringing the membranes very close
3) Membrane lying close to each other fuse together

Question 37

What mechanism allows for recycling of the SNARE proteins?

Answer 37

The activity of NSF ATPase with available ATP dismantles the sic-SNARE complex and takes away the vSNARE for the future use

Question 38

What is happening randomly to ER resident proteins?

Answer 38

They are randomly transported to the Golgi via non-selective COPII vesicles.

Question 39

What is the mechanism of retrieval of free-floating ER proteins?

Answer 39

These proteins have a KDEL retrieval signal (Lys-Asp-Glu-Leu at the C-terminus) that binds to the KDEL receptor in Golgi, which “packs them” into COPI and transports back to the ER
KDEL receptor is also present in the ER but binds the retrieval signal only in Golgi and not in ER, because of the pH difference between the 2 compartments.

Question 40

What is the mechanism of retrieval of membrane-bound proteins?

Answer 40

These proteins have a retrieval signal (Lys-Lys-X-X at C-terminus) that is recognised by the coat proteins of COPI vesicle, which take them back to the ER

Question 41

What is the exocytosis pathway for the membrane-bound and secretion proteins? Which cells use this pathway?

Answer 41

The proteins travel from the ER via COPII to the Golgi -> through the Golgi (cis-, medial- and trans- parts of Golgi) -> are secreted from the Golgi to fuse with the plasma membrane in constitutive vesicles.
This pathway is used by all cells.

Question 42

What cells use hormone/NT/digestive enzyme exocytosis cells and how is it initiated?

Answer 42

Specialised cells in specialised tissues. It is initiated by a specific signal.

Question 43

How are proteins modified in the Golgi?

Answer 43

Each Golgi cisterna has a different set of enzymes that are responsible for differenet modifications.
The aspargine-bound sugars(added in the ER) are modified in different ways.
O-linked glycosylation - addition of sugar on serine and threonine.

Question 44

What are the 2 models of Golgi organisation and transport?

Answer 44

1) small transport vesicles move the proteins between the compartments of Golgi in both directions - old model
2) Compartmets of Golgi are not static but ‘mature’ into the next stage (cis Golgi network> cis>medial>trans>trans Golgi network) carrying the proteins with them, which is counterbalanced by a vesicular transport in the opposite direction. Maturation occurs through sending back all of the enzymes characteristic for each stage to the proceeding compartment via vesicular transport. - currently accepted model.

Question 45

What is the function of lysosome and what proteins are trafficked into it?

Answer 45

Lysosome is a degrading organelle, thus it needs to be supplied with proteases and lipases.

Question 46

How are proteins trafficked to the lysosome?

Answer 46

o Mannose added to protein in the ER -> Mannose phosphorylated in the Golgi
o Mannose 6-phosphate acts as a signal for transport to the late endosome -> Receptor in the TGN recognises M6P and packs it to the vesicles, which merge with late endosome
o Late endosome is acidic – ATPase pumps protons inside
o M6P dissociates from its receptor at low pH -> Phosphate is removed -> and the lysosomal hydrolase precursor will end up in the lysosome when they merge -> there it will activate at even lower pH and start degrading proteins

Question 47

What molecules are endocytosed in a LDL? What are they important for?

Answer 47

Cholesterol - important to build and maintain the cell membrane and produce the stroid hormones

Question 48

Describe the endocytosis mechanism of LDL.

Answer 48

Receptors for LDL are expressed on the cellular membrane. The LDL binds to its receptor, which has an endocytosis signal (-FDNPVY-) -> the adaptor proteins bind to the signal and concentrate the receptor-LDL complexes into the cathrin pits -> coated pit forms a vesicle -> vesicle uncoats and fuses with early endosome
In slightly acidic pH(endosome) the receptor and LDL disassociate -> the receptor is recycled and LDL is taken to the lysosome. In the lysosome the LDL is degraded and the free cholesterol is released.

Question 49

Why is EGF endocytosed with its receptor?

Answer 49

EGF endocytosis is important to turn of EGF signalling, which causes cell division and if not turned off it may cause cancer.

Question 50

Describe the endocytosis of tthe EGF and its receptor.

Answer 50

EGF binds to its receptor on the cellular membrane -> adaptor proteins bind to the signal (-FYRAL-) -> cytosolic part of the EGF receptor is ubiquitinated -> vesicle forms and fuses with early endosome -> both receptor and EGF stay in endosome and go into the late endosome, where they are invaginated into the multivesicular bodies (the EGF ends up inside the lysosomal matrix, not in the cytosol) -> ESCO proteins recognise the ubiquitin signal on the EGF receptor -> endosome fuses with lysosome and the EGF and EGF receptor are degraded
- General endocytosis signal – (-YXXØ-) Ø – any hydrophobic residue

Question 51

Describe how influenza enters the cell as an example of viral endocytosis.

Answer 51

influenza binds to cell-surface receptors -> gets taken up to the endosome -> when the endosome reaches a certain pH, its fusion particles will fuse with the endosome membrane and allow for the release of the virus.