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ESA 1 - Introduction to Cell Physiology and Pharmacology > Session 2.2b - Lecture 2 - Membranes: Biological Function > Flashcards

Session 2.2b - Lecture 2 - Membranes: Biological Function Flashcards

Slides 17 - 29

1
Q

How can we figure out which membrane proteins are peripheral and which are integral?

A

We can perform a salt wash - membranes that come off in the salt wash are peripheral and the ones that stay in the membrane are integral.

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2
Q

Which proteins are integral membrane proteins of erythrocyte membranes?

A

(Very few)
Band 3
Band 7
(there are other ones but the detail is not important, just the concept - that some proteins are integral and some are peripheral)

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3
Q

How do we know Band 3 and Band 7 are integral proteins?

A

They are not dislocated in a salt wash

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4
Q

Name a peripheral membrane protein of erythrocyte membranes.

A

Spectrin

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5
Q

How do we know Spectrin is a peripheral protein?

A

It is washed off in a salt wash

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6
Q

What type of protein is spectrin?

A

A (LARGE) peripheral protein - it is found on the surface of the membrane rather than stuck through it.

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7
Q

Fig. 17

What does this image show?

A

Cartoonised electrophoresis of an erythrocyte membrane before and after a salt wash. After a salt wash the membranes contain very few proteins.

Ghost Membranes shows all membrane proteins
Ghost membranes after salt wash shows membranes that haven’t been washed off i.e. integral membrane proteins
Salt wash shows proteins that have been dislocated in the salt wash and removed from the membrane i.e. peripheral membrane proteins

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8
Q

Fig. 17

Caption and label this image.

A

Peripheral and Integral Proteins of the Erythrocyte Membrane

SDS-PAGE Electrophoresis - --> +
1 2 3 4.1 4.2 5 6 7
Ghost Membranes
Ghost membranes after salt wash
Salt wash
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9
Q

Draw the results of an electrophoresis to determine peripheral and integral proteins of an erythrocyte membrane.

A

See Fig. 17

Peripheral and Integral Proteins of the Erythrocyte Membrane

SDS-PAGE Electrophoresis - --> +
1 2 3 4.1 4.2 5 6 7
Ghost Membranes
Ghost membranes after salt wash
Salt wash
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10
Q

How can we visualise spectrin on an EM grid?

A
  • Take spectrin
  • Purify it (don’t need to know how)
  • Use electron-dense ion like osmium
  • Build up osmium against the molecule, like a snow drift
  • This creates an electron density of the ‘snow drift’
  • Which creates a low-angle shadow so we can see the image
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11
Q

What is the structure of spectrin?

A

Pairs of spectrin molecules winding around each other to form coiled-coiled molecule

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12
Q

Fig. 17+

Caption this image and add the scale bar.

A

Shadowed spectrin molecules

100 nm

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13
Q

Draw an electron micrograph of spectrin.

A

See Fig. 17+

Shadowed spectrin molecules

Scale bar: 100 nm (diameter of spectrin)

Pairs of spectrin molecules winding around each other to form coiled-coiled molecule

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14
Q

How can we visualise images using an electron microscope?

A
  • Use electron-dense ion such as osmium

- This builds up a ‘snow drift’ against molecules so structures can be visualised

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15
Q

How many molecules of spectrin are there winding around each other?

A

2

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16
Q

How do spectrin molecules interact?

A

Wind around each other forming a coiled-coiled molecule.

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17
Q

Fig. 17++

What is this image showing?

A

Cartoonised structure of spectrin - two spectrin molecules winding around each other

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18
Q

How does the structure of spectrin relate to its properties?

A

It has two spectrin molecules winding around each other which will create quite a strong rod-like structure.

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19
Q

Spectrin is created by two spectrin molecules winding around each other.

What property will this give spectrin?

A

A strong rod-like structure.

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20
Q

How do the properties of spectrin relate to its function?

A

Two spectrin molecules winding around each other, which will create quite a strong rod-like structure - which would be quite a good unit to put into some sort of cage within a cell.

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21
Q

Spectrin has a strong rod-like structure - how does this relate to function?

A

This would be quite a good unit to put into some sort of cage within a cell.

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22
Q

Fig. 17++

Label and caption the image.

A

a CHAIN
H2N
- flexible link between domains
COOH

b CHAIN
HOOC
4 Ps
- 106-amino-acid-long domain
NH2
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23
Q

Draw a spectrin molecule (5 marks)

A

See Fig. 17++

a CHAIN
H2N
- flexible link between domains
COOH

b CHAIN
HOOC
4 Ps
- 106-amino-acid-long domain
NH2
(- 2 chains
- 106 amino acids long
- NH2 - COOH reversed on other side
- b chain has 4 Phosphates on COOH side
- flexible link between domains
5 marks)
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24
Q

How can we visualise the cytoplasmic face of an erythrocyte membrane?

A

So let’s do another experiment, let’s take an erythrocyte and look at cytoplasmic face of erythrocyte membrane.

So again what we’ll do is open our erythrocyte, shadow at low-angle with osmium, look under EM

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25
Q

What do we see when we look at the cytoplasmic side of an erythrocyte membrane?

A

We can see lattice, cage-like structures attached to inside face of erythrocyte membranes

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26
Q

What are the rod-like structures we see in the cytoplasmic face of the erythrocyte membrane?

A

Spectrin molecules

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27
Q

What do spectrin molecules look like?

A

Rod-like structures

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28
Q

Where are spectrin molecules found?

A

The surface of the cytoplasmic face of the erythrocyte membrane.

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29
Q

What is the layout of spectrin molecules?

A

They lie end-to-end, i.e. two of those coiled-coils lie end-to-end.

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30
Q

Spectrin hasn’t been washed off when we open the inside of our erythrocyte membrane up. What does this mean?

A

It must be linked through the membrane through integral membrane proteins – there must be points of attachment where these coiled-coils are in some ways glued to the transmembrane proteins to keep that lattice around the inside face of the erythrocyte

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31
Q

How does spectrin stay as a lattice on the inside face of the erythrocyte?

A

There must be points of attachment where these coiled-coils are in some way glued to the transmembrane protein.

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32
Q

Fig. 17+++

Label and caption this image.

A

Negatively stained erythrocyte cytoskeleton

  • spectrin
  • ankyrin
  • actin in junctional complex
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33
Q

Draw an erythrocyte cytoskeleton.

A

See Fig. 17+++

Negatively stained erythrocyte cytoskeleton

  • spectrin (perimeters of lattice)
  • ankyrin (dark patches on spectrin)
  • actin in junctional complex (at corners)
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34
Q

What does spectrin do in the erythrocyte membrane?

A

Spectrin dimers form a lattice with transmembrane proteins, making an interaction through proteins.

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35
Q

Fig. 17d

Label and caption this image.

A

Spectrin-based cytoskeleton

  • junctional complex
  • spectrin dimer
  • actin
  • ankyrin
  • band 3
  • glycophorin
  • band 4.1
    [100 nm]
  • ->
  • adducin
  • actin
  • band 4.1
  • tropomyosin
  • spectrin
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36
Q

Draw the cytoskeleton of an erythrocyte membrane, labelling the proteins.

A

See Fig. 17d

Spectrin-based cytoskeleton

  • junctional complex
  • spectrin dimer
  • actin
  • ankyrin
  • band 3
  • glycophorin
  • band 4.1
    [100 nm]
  • ->
  • adducin
  • actin
  • band 4.1
  • tropomyosin
  • spectrin
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37
Q

Describe the cytoskeleton of the erythrocyte.

A

Lattice of spectrin coiled-coils lining up head-to-tail in the membrane to form a lattice

Learn

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38
Q

How does spectrin interacting with the bilayer?

A

Through

  • Band 3 (important protein)
  • Band 7
  • Glycophorin
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39
Q

Name an anchoring protein.

A

Ankyrin

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40
Q

What does ankyrin do?

A
  • it is an ANCHORING protein

- forms an interaction between BAND 3 and SPECTRIN

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41
Q

Other than Band 3 and ankyrin, which other proteins interact to anchor spectrin to the bilayer?

A
  • Band 4.1
  • Actin
  • Adducin
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42
Q

How are Band 3, Band 4.1 and Band 7 etc. so named?

A

This is their numbering within electrophoresis

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43
Q

What is actin?

A

A muscle protein

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44
Q

What is adducing?

A

Another gluing protein

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45
Q

What does the spectrin lattice do to integral proteins?

A

Fixes them, restricting their motion

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46
Q

What is the model of the simplest cytoskeleton?

A

Erythrocyte membrane:

Lattice under the membrane glued on by these adaptor proteins - holding the lattice against TM proteins.

Do not need to know what the individual proteins do and where it is in that structure

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47
Q

Describe the general structure of the cytoskeleton

Possible exam q

A

Rod-like proteins glued onto the membrane

Level you need to know - do not need to know what the individual proteins do and where it is in that structure

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48
Q

What are the cytoskeleton of other cells (compared to the erythrocyte membrane) like?

A

Other cells have a cytoskeleton and they have other proteins that are involved e.g. spectrin-like proteins like fodrin etc., so they are more complex than the erythrocyte skeleton.

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49
Q

Give an example of other cytoskeletal proteins.

A

Spectrin-like proteins, e.g. fodrin

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50
Q

What is the function of the cytoskeleton?

A

It puts a cage-like structure around the inside surface of the cell membrane.

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51
Q

What is the function of the cytoskeleton for the erythrocytes?

A

Puts a cage-like structure around inside surface of the cell membrane so when the cell is pushing through capillary the cell is able to bend and change shape but maintain its integrity as it goes through the capillary.

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52
Q

Fig. 18

Caption and label this image.

A

Erythrocyte cytoskeleton

Extracytoplasmic surface (outside)
Membrane (grey line)
Cytoplasmic surface (with spectrin)
Band 3 (green circle)
Glycophorin A (green rectangle)
Ankyrin (band 4.9) (blue circle)
Band 4.1 (pink circle)
a b Spectrin (chains)
Actin (purple circles)
Band 4.1 (pink circle)
Adducin (arrows)
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53
Q

Draw the erythrocyte cytoskeleton.

A

See Fig. 18

Erythrocyte cytoskeleton

Extracytoplasmic surface (outside)
Membrane (grey line)
Cytoplasmic surface (with spectrin)
Band 3 (green circle)
Glycophorin A (green rectangle)
Ankyrin (band 4.9) (blue circle)
Band 4.1 (pink circle)
a b Spectrin (chains)
Actin (purple circles)
Band 4.1 (pink circle)
Adducin (arrows)

Do not need to know all this detail

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54
Q

What is the function of these proteins and where are they? (Do not need to know this level of detail)

  • Actin
  • Adducin
  • Ankyrin (band 4.9)
  • Band 3
  • Band 4.1
  • Glycophorin A
  • Spectrin
A
  • Actin: anchoring protein, interacts with Band 4.1 and Adducin on Spectrin
  • Adducin: at ends of Spectrin, interacts with Actin and Band 4.1
  • Ankyrin (band 4.9): anchoring protein between Band 3 and Spectrin
  • Band 3: transmembrane protein
  • Band 4.1: anchoring protein between Glycophorin and Spectrin
  • Glycophorin A: transmembrane protein
  • Spectrin: rod-like lattice protein.
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55
Q

Name two types of haemolytic anaemias that are caused by defects in spectrin.

A
  • Hereditary spherocytosis

- Hereditary elliptocytosis

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56
Q

Give 4 features of hereditary spherocytosis.

A

– Spectrin depleted by 40-50%
– Erythrocytes round up
– Less resistant to lysis
– Cleared by spleen

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57
Q

Give 3 features of hereditary elliptocytosis.

A

– Defect in spectrin molecule
– Unable to form heterotetramers
– Fragile elliptoid cells

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58
Q

What is the epidemiology of hereditary spherocytosis?

A

Rare

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59
Q

What is the pathophysiology for hereditary spherocytosis?

A

Spectrin expression is reduced – you can have two alleles for spectrin, if one is mutated, now no longer making spectrin off that allele we’ll be getting half as much RNA as we had before so we’ll have half the amount of protein we had before, leading to a weak cytoskeleton.

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60
Q

How can spectrin expression be reduced, e.g. in hereditary spherocytosis?

A

By a mutation in the allele

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61
Q

What would a mutation in the spectrin allele lead to?

A

A reduction in the RNA for spectrin produced, therefore a reduction in the overall protein produced.

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62
Q

What does a mutation in the spectrin allele lead to functionally?

A

As it’s a structural protein we’ll only have half the normal amount of structural protein so what you end up with are erythrocyte cytoskeleton that form but not properly formed because they don’t have all the components they need, so they have a weak cytoskeleton - this is known as hereditary spherocytosis.

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63
Q

How does hereditary spherocytosis lead to anaemia?

A

In hereditary spherocytosis, there is a mutation in one of the spectrin alleles, leading to reduced protein expression in spectrin. This means that the cytoskeleton doesn’t form properly, and it is WEAK. This means the biconcave shape of the disc is not always maintained, so as the RBC passes through the capillary it has the potential to shear, releasing the Hb, resulting in the pt becoming anaemic.

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64
Q

Why is hereditary spherocytosis so named?

A

Hereditary - genetic; defective allele

Spherocytosis - cells round up and form spherical cells (rather than biconcave flexible discs), and lyse.

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65
Q

What clears the cells in hereditary spherocytosis?

A

They are particularly cleared by the spleen, which has a dense network of small capillaries.

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66
Q

What is the pathophysiology of hereditary elliptocytosis?

A

Both alleles express spectrin but one of these genes in unable to form head-to-tail associations with its partner, so you end up with plenty of spectrin but all misformed in cytoskeleton structure. These are unable to form the rod-like structures within the lattice and end up with fragile cells

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67
Q

If spectrin cannot form head-to-tail, what does this lead to functionally?

A

Unable to form the rod-like structures within the lattice and end up with fragile cells

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68
Q

Why is hereditary elliptocytosis so named?

A

Hereditary - genetic; defective allele

Elliptocyotsis - cells end up elliptoid (like a rugby ball).

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69
Q

What is the epidemiology of hereditary elliptocytosis?

A

Rare

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70
Q

What is the treatment option for hereditary spherocytosis?

A

Blood transfusion

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71
Q

What is the treatment option for hereditary elliptocytosis?

A

Blood transfusion

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72
Q

Why is the treatment option for hereditary spherocytosis and elliptocytosis blood transfusion?

A

Hereditary – always going to be making these spectrins, so only thing you can do for a patient in anaemic crisis is give them a blood transfusion of normal blood with normal spectrin, normal biconcave cells to give them some days of oxygen carrying capacity

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73
Q

When is a blood transfusion given to a patient with hereditary spherocytosis/elliptocytosis?

A

When they are in anaemic crisis

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74
Q

What does giving a blood transfusion do to a patient with hereditary spherocytosis/elliptocytosis?

A

Gives them a blood transfusion of normal blood with normal spectrin, normal biconcave cells to give them some days of oxygen carrying capacity

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75
Q

What is the impact on quality of life/prognosis for patients with hereditary spherocytosis/elliptocytosis?

A

Patients must live with a debilitating condition for the whole of their life

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76
Q

What are hereditary spherocytosis/elliptocytosis caused by?

A

Simple mutations in their cytoskeleton (spectrin)

77
Q

Compare and contrast:

  • Normal patients
  • Hereditary spherocytosis
  • Hereditary elliptocytosis

(Epidemiology, RBC Shape, Pathophysiology and Consequences)

A

Normal patients:

  • Epidemiology: normal/common
  • RBC shape: biconcave flexible disc
  • Pathophysiology: none - two spectrin dimers intact to form cytoskeleton
  • Consequence: RBCs able to fit through small capillaries

Hereditary spherocytosis:

  • Epidemiology: rare
  • RBC shape: round up to form spheres
  • Pathophysiology: mutation in one spectrin gene so not enough protein is produced and therefore cytoskeleton is weak (depleted by about 40-50%)
  • Consequence: RBCs lyse when fitting through small capillaries

Hereditary elliptocytosis:

  • Epidemiology: rare
  • RBC shape: elliptoid (rugby ball)
  • Pathophysiology: mutation in spectrin gene so that spectrin is produced but is misformed - so lattice is unable to form
  • Consequence: spectrin unable to form heterotetramers so RBC is fragile
78
Q

If we can’t flip flop protein through a membrane, how do we get that hydrophilic part of the protein through the membrane so that the protein ends up as seen in the Singer-Nicholson model?

A

Via the same mechanism as secreted protein biosynthesis; but it is made/stays in the membrane

  • Membrane protein could use the same targeting mechanism, it could express signal, be arrested by SRP, be brought down to ER attached to docking protein, send N-terminal through translocational machinery, in that case, the protein would still be continued to synthesised
79
Q

How are secreted proteins, such as insulin, made?

A
  • DNA transcription to RNA in the nucleus of the cell
  • RNA leaves nucleus into the cytoplasm and is recognised by ribosomes
  • Ribosomes secrete a 18-20AA hydrophobic sequence at the N-terminal of the protein, called the SIGNAL SEQUENCE (SS)
  • SS is recognised by a SIGNAL RECOGNITION PARTICLE (SRP)
  • SRP binds both SS and ribosome together, locking the nascent protein and arresting synthesis in the cytoplasm
  • SRP brings associated SS and ribosome down to the endoplasmic reticulum (ER) membrane, where it binds to a DOCKING PROTEIN (DP)
  • SRP lands on DP, whilst the SS is recognised by a signal sequence receptor (SSR).
  • The SS and ribosome unbind from the SRP/DP to bind to the SSR
  • This then allows protein synthesis to occur through a protein translocator complex (PTC) in the membrane
  • Protein is therefore synthesised in the ER lumen and ready to be packaged into vesicles
  • A SIGNAL PEPTIDASE (SP) cuts off the SS in the lumen, as it is no longer needed there
80
Q

Where does insulin need to enter before its release?

A

So what we want to do really is while we’re making insulin at the same time make sure that it enters some membrane compartment, so when it’s fully formed it can be sent to the surface for release.

81
Q

Where does insulin need to be released from?

A

Not in the cytoplasm but in a vesicle that can be released from the cell, because it is a secreted protein

82
Q

Where does insulin need to enter before its release if it needs to be in a vesicle?

A

So what we want to do really is while we’re making insulin at the same time make sure that it enters some membrane compartment, so when it’s fully formed it can be sent to the surface for release.

83
Q

How does the genetic code become RNA?

A

Gene in DNA is TRANSCRIBED into RNA.

84
Q

Where is RNA?

A

DNA is transcribed into RNA in the NUCLEUS, where it then leaves to the CYTOPLASM.

85
Q

What reads the RNA code?

A

Macromolecules known as RIBOSOMES

86
Q

What do ribosomes read?

A

The TRIPLET CODE of the RNA structure.

87
Q

How is RNA made into proteins?

A

RNA is TRANSLATED by ribosomes in the cytoplasm to form the protein sequence.

88
Q

What do ribosomes create?

A

The PRIMARY SEQUENCE of the PROTEIN.

89
Q

What is the problem of making insulin via normal protein synthesis methods?

A

If that process happened and we made insulin we’d have insulin in our cytoplasm, then we would have a problem now bc we would have to select it from all that mash of stuff and package it for release

90
Q

Give an example of a secreted protein.

A

Insulin

91
Q

Secreted proteins, such as insulin do what to their protein?

A

As they’re made, at their N –terminal end, they have a highly hydrophobic sequence about 18-20 AAs

92
Q

Secreted proteins, such as insulin do what to their protein?

A

As they’re made, at their N –terminal end, they have a highly hydrophobic sequence about 18-20 AAs - signal sequence.

93
Q

Where is the signal sequence of secreted proteins, e.g. insulin?

A

On their N-terminal end

94
Q

What are signal sequences?

A

Highly hydrophobic sequences about 18-20 AAs long, on the N-terminal end of the protein.

95
Q

How are signal sequences made?

A

As the ribosome starts making a secreted protein (e.g. insulin) the first thing that comes out of the ribosome into the cytoplasm is this hydrophobic sequence, and we call that hydrophobic sequence a signal sequence

96
Q

What is the signal sequence recognised by?

A

The signal recognition particle (SRP).

97
Q

What is the signal recognition particle (SRP)?

A

An RNA-protein complex: a big macromolecular complex that recognises the signal sequence.

98
Q

What do signal recognition particles (SRPs) bind to?

A

The signal sequence AND the ribosome

99
Q

What is the function of the signal recognition particle (SRP)?

A
  • To lock the nascent (new) protein being synthesised from the ribosome against it - it does this by binding to both the signal sequence and ribosome - thus ARRESTING SYNTHESIS in the cytoplasm.

It also brings this signal sequence to the endoplasmic reticulum.

100
Q

How is the ribosome prevented from translating further amino acids after the signal sequence is produced?

A

Signal recognition particle (SRP) locks the signal sequence against the ribosome by binding to both.

So now although the ribosome might be trying to read further AAs, it can’t push them through bc the whole thing has been locked up by the SRP

101
Q

Where do signal recognition particle (SRP) act?

A

In the cytoplasm (on the signal sequence and ribosome) - so further translation does not occur in the ribosome.

102
Q

How does the signal recognition particle (SRP) get to the endoplasmic reticulum (ER) in a protein making insulin?

A

So now the SRP can come down to the endoplasmic reticulum, where it sees a docking protein, so the SRP is holding a ribosome, and it comes down to the ER where it sees a docking protein, bringing that particular ribosome, making insulin, down to the ER

103
Q

What is found on the ER membrane that binds to the SRP?

A

Docking protein

104
Q

What happens to the SRP and its associated complex once it reaches the ER?

A

When it gets to ER, signal is released by SRP (on the docking protein), and is bound by a signal sequence receptor (SSR).

105
Q

Where are signal sequence receptors (SSR) found?

A

Within the bilayer of the ER.

106
Q

Once on the SSR, how does the protein sequence of secreted proteins get into the ER?

A

The signal is fed through a protein translocator complex (Sec61), into the lumen of the ER

107
Q

What is the function of the SSR/protein translocator complex?

A

To feed the signal sequence and protein through the ER membrane into the lumen of the ER.

108
Q

What does the docking protein recognise?

A

Signal recognition particle (SRP).

109
Q

What does a signal sequence do?

A

Acts as a signal to arrest protein synthesis in the cytoplasm and binds to SSRs to allow synthesis to continue in the ER lumen.

110
Q

Once the ribosome is on the ER membrane, how does synthesis occur?

A

So we arrested synthesis, brought it down to ER, signal now recognised by translocation machinery, allows then the ribosome to keep synthesising, but now the ribosome is sitting on the moon and sending the new strand into lumen of ER so when synthesis is finished the whole thing is in the lumen of the ER, so it is packaged already

111
Q

What recognises the signal sequence on the ER membrane to allow protein synthesis to occur?

A

Translocation machinery which allows the ribosome to keep synthesising

112
Q

Where does the ribosome act in secreted protein biosynthesis?

A
  • Makes signal sequence in cytoplasm and is then arrested
  • When brought down to ER membrane, sends new strand through protein translocator complex in the membrane into the lumen of the ER.
113
Q

What happens to the signal sequence in the ER lumen?

A

The hydrophobic sequence is chopped off

114
Q

What chops off the signal sequence in the ER lumen?

A

Signal peptidase

115
Q

What is the function of signal peptidase?

A

To chop off the signal sequence in the ER lumen because it is no longer needed

this is important

116
Q

What is the structure of insulin?

A

It has an a and b chain.

117
Q

How is insulin made (structurally)?

A

As a pro-peptide

118
Q

Insulin is produced as a pro-peptide. What occurs to it to create its final form.

A

Post-translational modification.

119
Q

What is the post-translational modification that occurs?

A

Made as a pro-peptide and cleaved into 2 polypeptides (a and b chain)

120
Q

Describe the function of

  • Signal recognition particle (SRP)
  • Docking protein (DP)
  • Signal sequence (SS)
  • Signal sequence receptor (SRP) / protein translocator complex (PTC)
  • Signal peptidase (SP)
A

SRP: locks SS to ribosome, arresting synthesis in cytoplasm and brings nascent protein/ribosome complex down to the ER membrane.

DP: found on ER membrane which allows SRP to bind to and release the SS/ribosome complex.

SS: signals to ribosome to stop making protein synthesis in the cytoplasm -binds to SRP

SSR/PTC: found on ER membrane, binds to SS which allows protein synthesis to recontinue in the lumen of the ER

SP: cleaves off SS in ER lumen as no longer needed

121
Q

Where are the following found?

  • Signal recognition particle (SRP)
  • Docking protein (DP)
  • Signal sequence (SS)
  • Signal sequence receptor (SRP) / protein translocator complex (PTC)
  • Signal peptidase (SP)
A

SRP: cytoplasm

DP: ER lumen membrane (facing cytoplasm)

SS: N-terminus of nascent protein (cytoplasm then travels to ER lumen to be cleaved)

SSR/PTC: ER lumen membrane (facing cytoplasm)

SP: ER lumen

122
Q

Where are the following found?

  • Signal recognition particle (SRP)
  • Docking protein (DP)
  • Signal sequence (SS)
  • Signal sequence receptor (SRP) / protein translocator complex (PTC)
  • Signal peptidase (SP)
A

SRP: cytoplasm

DP: ER lumen membrane (facing cytoplasm)

SS: N-terminus of nascent protein (cytoplasm then travels to ER lumen to be cleaved)

SSR/PTC: ER lumen membrane (facing cytoplasm)

SP: ER lumen

123
Q

Fig. 20

Caption and label this image.

A

Secreted Protein Biosynthesis

Cytoplasm
ER lumen
5’ to 3;

Key:

  • SRP
  • Docking protein
  • Signal sequence
  • Signal sequence receptor / protein translocator complex (Sec61)
  • Signal Peptidase
124
Q

Draw and label the pathway for secreted protein biosynthesis.

A

See Fig. 20

Secreted Protein Biosynthesis

Cytoplasm
ER lumen
5’ to 3;

Key:

  • SRP
  • Docking protein
  • Signal sequence
  • Signal sequence receptor / protein translocator complex (Sec61)
  • Signal Peptidase

(See Notability for explanation)

125
Q

Signal sequences of secreted proteins contain hydrophobic sequences of 18-20 AAs.

How can this be used in membrane protein synthesis?

A

Membrane protein transmembrane domains have hydrophobic runs of about 20-22 AAs that make up their alpha helix. This means, when a membrane protein is being synthesised, the hydrophobic portion is made and stays in the membrane rather than gets extruded into the lumen.

126
Q

Transmembrane proteins are locked into the membrane via their hydrophobic domains. What is this called?

A

Stop-transfer sequence.

127
Q

What does the stop-transfer sequence do?

A

It is the hydrophobic domain of a transmembrane protein that runs through the membrane, preventing this portion from being extruded out into the lumen.

It stops the protein in the membrane.

128
Q

Where is the N-terminus and C-terminus of a membrane usually found?

A

N-terminal - facing lumen

C-terminal - facing cytoplasm

129
Q

Why are most transmembrane proteins N-terminal lumen side and C-terminal cytoplasmic side?

A

Bc of the way the protein is made - the ribosome is still working away trying to make protein so lives off ER, & makes rest of protein in cytoplasm. So end up with protein that is transmembranous with its N-terminal facing the lumen, C-terminal facing cytoplasm.

130
Q

How are transmembrane proteins biosynthesised?

A
  • Using the same mechanism that secreted protein synthesis to the ER uses, whereby the signal sequence is a hydrophobic sequence that arrests synthesis in the cytoplasm
  • This however, occurs in the protein sequence, where the transmembrane domain is a hydrophobic portion, keeping this in the membrane.
  • This hydrophobic portion is called the stop-transfer sequence
131
Q

How does the stop-transfer sequence create a transmembrane protein?

A

The stop-transfer sequence is a hydrophobic sequence that is made, which just doesn’t thermodynamically want to be passed through the membrane, so locks the protein in the membrane and causes that protein to be completed as a transmembrane protein

132
Q

How is a transmembrane protein with an N-terminal facing lumen created?

A
  • Secreted protein synthesis machinery is used
  • Ribosome on membrane of, e.g. the ER, synthesises protein through the membrane
  • Extrudes protein into the lumen
  • Stops when it gets to the stop-transfer sequence (hydrophobic patch)
  • Continues making rest of protein in the cytoplasm
  • Therefore N-terminal facing lumen but C-terminal facing cytoplasm
133
Q

Give an example of a protein with a C-terminal facing the lumen.

A

Glycophorin (erythrocyte membrane protein).

134
Q

Fig. 21

Caption and label the protein.

A

Membrane Protein Biosynthesis

Cytoplasm
ER lumen
3’

Key:

  • SRP
  • Docking protein
  • Signal sequence
  • Signal sequence receptor / ribophorins
  • Signal Peptidase
135
Q

Draw the pathway of membrane protein biosynthesis.

A

See Fig. 21

Membrane Protein Biosynthesis

Cytoplasm
ER lumen
3’

Key:

  • SRP
  • Docking protein
  • Signal sequence
  • Signal sequence receptor / ribophorins
  • Signal Peptidase
136
Q

What is Sec61?

A

Sec61 is an endoplasmic reticulum (ER) membrane protein translocator (aka translocon). It is a doughnut-shaped pore through the membrane with 3 major subunits (heterotrimeric). It has a region called the plug that blocks transport into or out of the ER. This plug is displaced when the hydrophobic region of a nascent polypeptide interacts with another region of Sec61 called the seam, allowing translocation of the polypeptide into the ER lumen. [Wikipedia]

137
Q

What are ribophorins?

A

They are glycoproteins found on the RER membrane which play a role in co-translational translocation of proteins into the cytoplasm into the ER lumen.

138
Q

For interest only

What happens to the N-terminus signal sequence when it passes through the membrane?

A

It actually has a few basic +ve charged residues at the N-terminus, so the last thing it wants to d

139
Q

What happens to the N-terminus signal sequence when it passes through the membrane?

For interest only

A

It actually has a few basic +ve charged residues at the N-terminus, so the last thing it wants to do is go head first into the ER

140
Q

What way would a transmembrane protein want to bind thermodynamically?

For interest only

A

Actually wants to bind with N-terminus on cytoplasmic side and C-terminal with signal sequence of luminal side.

141
Q

What happens to the N-terminus signal sequence in transmembrane proteins?

(For interest only)

A

Signal peptidase is still in the right place to cleave the signal peptidase cleavage site, releasing the N-teminus

142
Q

How does an N-terminal signal sequence transmembrane protein get its orientation?

For interest only

A
  • N-terminus with basic +ve signal sequence enters the membrane from the cytoplasmic side
  • This ‘flops back’, but the signal peptidase cleavage site is still on the ER lumen side
  • So N-terminal end is still in the ER lumen with C-terminal end of cytoplasmic side
143
Q

What is different about the signal sequence in an N-terminal transmembrane protein and a secreted protein biosynthesis?

(For interest only)

A
  • Secreted protein biosynthesis: goes into ER lumen and is cleaved
  • N-terminal transmembrane protein: flops into ER membrane and back out, but is still cleaved in ER lumen
144
Q

For interest only
Fig. 22

Label and caption this image.

A

Membrane protein orientation
Secretion into ER lumen
(N-terminal signal sequence)

N++
Signal peptidase

N++
Cytoplasm
ER lumen
N
releases N-terminal into the ER lumen
145
Q

For interest only

Draw the membrane protein orientation for N-terminal lumen signal sequence.

A

See Fig. 22

Membrane protein orientation
Secretion into ER lumen
(N-terminal signal sequence)

N++
Signal peptidase

N++
Cytoplasm
ER lumen
N
releases N-terminal into the ER lumen
146
Q

For interest only

Fig. 23

Explain what this image is showing.

A

So our membrane protein then can flop in, can be cleaved, the synthesis can continue, the stop-transfer sequence can stop the protein in the membrane and the C-terminal can be completed in the cytoplasm: the model works fine.

147
Q

For interest only

Fig. 23

Caption and label this image.

A

Membrane protein orientation
N-terminal, cleavable signal sequence
N-terminal to ER lumen

N ++
Signal peptidase

N ++
N

C
Cytoplasm
ER Lumen
N

148
Q

For interest only

Draw a transmembrane protein synthesis with N-terminal facing the lumen and C-terminal facing the cytoplasm.

A

See Fig. 23

Membrane protein orientation
N-terminal, cleavable signal sequence
N-terminal to ER lumen

N ++
Signal peptidase

N ++
N

C
Cytoplasm
ER Lumen
N

149
Q

For interest only

If there was no signal peptidase cleavage site at the end of the signal sequence in a transmembrane protein, what would happen?

A

There would be no cleavage

150
Q

For interest only

If there is no cleavage of the signal sequence in a transmembrane protein, what would be the end result?

A

The ribosome would still keep making the protein, it would now make it as a growing loop of the protein, that if we allowed synthesis to continue that would result with the N terminal now facing out into the cytoplasm and the C terminal into the lumen of the ER

151
Q

For interest only

What would be the result of the ribosome continuing to make the protein without signal peptidase cleavage?

A

If we allowed synthesis to continue that would result with the N terminal now facing out into the cytoplasm and the C terminal into the lumen of the ER

152
Q

For interest only

What would happen in the presence of an N-terminal signal sequence in the absence of a signal peptidase cleavage site?

A

C-terminal to ER lumen

  • There would be no signal sequence cleavage, so the ribosome would still keep making the protein, it would now make it as a growing loop of the protein, that if we allowed synthesis to continue that would result with the N-terminal now facing out into the cytoplasm and the C-terminal into the lumen of the ER
153
Q

For interest only

Fig. 24

Caption and label this image.

A

Membrane protein orientation
What would happen in the presence of an N-terminal signal sequence in the absence of a signal peptidase cleavage site?
C-terminal to ER lumen

N++
No signal peptidase

N++

N
Cytoplasm
ER Lumen
C

154
Q

For interest only

Draw a diagram depicting what would occur if there was a signal sequence but no signal peptidase.

A

See Fig. 24

Membrane protein orientation
What would happen in the presence of an N-terminal signal sequence in the absence of a signal peptidase cleavage site?
C-terminal to ER lumen

N++
No signal peptidase

N++

N
Cytoplasm
ER Lumen
C

155
Q

For interest only

How can we subvert the N-terminal no signal peptidase model into a realistic structure?

A

The N-terminal hydrophilic protein could be being made as if it was a cytoplasmic protein.

156
Q

For interest only

How can we get the transmembrane domain in the membrane?

A

Ribosomes could be making the protein and then suddenly it comes and forms an internal signal in the primary sequence, so the first TM domain if you like, could now be within the body of the protein

157
Q

For interest only

What is different about the signal sequence and the sequence in a transmembrane domain?

A

Signal sequence: at the start of a protein (and usually cleaved by signal peptidase)

Transmembrane domain: signal is within the body of the protein (i.e. in the middle of the primary sequence)

158
Q

For interest only

How do we get the N-terminal sequence on the cytoplasmic side?

A

So we’ve made some, then signal is made, SRP system sees that and brings it down into the membrane, that can work bc all that N-terminal sequence where it was just 2 basic residues before (could now be a whole chunk of protein, doesn’t matter) can flop into membrane as before, no peptidase cleavage so loop continue to form, end up with protein reversed in orientation

159
Q

For interest only

What is the main functional difference in getting the N-terminal end of a protein on the cytoplasmic side?

A

There is NO signal peptidase, so instead of the signal sequence being cleaved and the N-terminal being on the lumenal side, and the C-terminus continuing to grow on the cytoplasmic side - instead, the signal sequence is not cleaved so the protein loops around and keeps growing but is reversed in orientation, so the N-terminus is on the cytoplasmic side.

160
Q

For interest only

Proteins that have a N-terminal signal sequence will what?

A

Have their N-terminus facing the lumen bc they all have a peptidase cleavage site.

161
Q

For interest only

Proteins that have an internal signal sequence will what?

A

The proteins that have an internal signal sequence – buried somewhere in primary sequence - will end up being switched around bc the early form N-terminal protein can’t go across the membrane but the signal can still flop in according to our model.

162
Q

For interest only

What is the significance in the location of the signal sequence?

A
  • proteins that have an N-terminal signal sequence will end up with their N-terminus facing their lumen bc they all have a peptidase cleavage site.
  • proteins that have an internal signal sequence – buried somewhere in primary sequence - will end up being switched around bc the early form N-terminal protein can’t go across the membrane but the signal can still flop in according to our model.
163
Q

For interest only

Fig. 25

Caption and label the image.

A

Membrane protein orientation
Start-transfer sequence contained within the primary sequence, positive charges at the N-terminal end?
C-terminal to ER lumen

N++
No signal peptidase

N++

N
Cytoplasm
ER Lumen
C

164
Q

For interest only

Draw the mechanism for how a protein can face N-terminally into the cytoplasm and C-terminally into the lumen.

A

Membrane protein orientation
Start-transfer sequence contained within the primary sequence, positive charges at the N-terminal end?
C-terminal to ER lumen

N++
No signal peptidase

N++

N
Cytoplasm
ER Lumen
C

165
Q

For interest only

Fig. 26

Caption and label the image.

(Slide skipped as don’t need to know)

A

Membrane protein orientation
Start-transfer sequence contained within the primary sequence, positive charges at the C-terminal end?
N-terminal to ER lumen

Ribosome continues synthesis in cytoplasm

C++
N
No signal peptidase

C
Cytoplasm
ER Lumen
N

166
Q

For interest only

Draw an image depicting what would happen if the start-transfer sequence contained within the primary sequence, positive charges at the C-terminal end?

A

See Fig. 26

Membrane protein orientation
Start-transfer sequence contained within the primary sequence, positive charges at the C-terminal end?
N-terminal to ER lumen

Ribosome continues synthesis in cytoplasm

C++
N
No signal peptidase

C
Cytoplasm
ER Lumen
N

(Slide skipped as don’t need to know)

167
Q

For interest only

What do the bacteria rhodopsin and GPCRs have in common?

A

They have multiple (7) transmembrane domains - they loop in and out.

168
Q

For interest only

How do you get a protein with multiple transmembrane domains, e.g. bacteria rhodopsin/GPCRs into the membrane?

A

WE ARE UNSURE

  • First TM domain is made as normal
  • Second TM domain could be another stop-transfer sequence, arresting further translocation

Current theory suggests that another pair is made like this and then ‘plugged in’ and joined - but we don’t know.

169
Q

For interest only

How can we get a protein with two transmembrane domains in?

A

You could imagine you could have a signal sequence flopping into the membrane, a loop forming, and then a second TM domain could be effectively a stop-transfer sequence – could arrest further translocation of that protein through the membrane – so now we have 2 TM domains in.

170
Q

For interest only

How can we get a protein with 3+ TM domains in?

A

So the 3rd could be another signal, could be as ribosome lifts off and starts making the next TM domain, SRP sees it and brings it back to the membrane.

OR

Alternatively, what could happen, we could make a cassette of two new TM domains and bring that whole chunk back – sort of plug it into the membrane.

171
Q

For interest only

What are the two models for multiple TM domain protein synthesis?

A

Each TM domain acts as a stop-transfer sequence and subsequent TM domains arrested by SRP

OR

TM domains are made in chunks of two and are rejoined back into the protein.

172
Q

For interest only

If the model was subsequent TM domains arrested by SRP and brought back, what would you see?

A

If you stopped the cell and looked at all the range of synthetic intermediates for our growing protein, you would see an even range of lengths of protein

173
Q

For interest only

If the model was protein needs to be formed in ‘cassettes’, and something needs to bring that cassette back, what would you see?

A

But if protein needs to be formed in cassettes, then something needs to happen to get that cassette back into the membrane then what you’d see is chunked sizes of protein

174
Q

For interest only

What is the evidence for the current form of multiple transmembrane protein synthesis?

A

If model was subsequent TM domains arrested by SRP and brought back, what you’d end up with, if you stopped the cell and looked at all the range of synthetic intermediates for our growing protein, you would see an even range of lengths of protein.

But if protein needs to be formed in cassettes, then something needs to happen to get that cassette back into the membrane then what you’d see is chunked sizes of protein and that’s what you see

175
Q

For interest only

What do we currently believe is the way multiple transmembrane targets are made?

A

So we’re not sure, but we believe proteins are targeted in the membrane in the same way as a single TM domain receptor protein, but then pairs of TM domains are made and plugged in

176
Q

What do I need to know about protein biosynthesis?

A

Need to be able to describe:

  • SRP docking cycle for insulin
  • SRP docking cycle for a single transmembrane protein, which is N-terminally directed to the lumen (just need to know early mechanism for getting protein in the membrane)
    (Slides 20 and 21)

DO NOT NEED TO KNOW DETAILS FOR C-LUMEN and MULTIPLE TRANSMEMBRANE (Slides 22 onwards)

177
Q

For interest only

Fig. 27

Label and caption the diagram.

A

Multiple spanning transmembrane proteins

e.g. G-protein coupled receptor

178
Q

For interest only

Draw a proposed diagram for how multiple spanning transmembrane proteins are produced.

A

See Fig. 27

Multiple spanning transmembrane proteins

e.g. G-protein coupled receptor

179
Q

How can we make a vesicle leaky?

A

By changing the osmotic strength

180
Q

How do we get an inside-out vesicle?

A

Well cells of PM is not static, it’s doing lots of things, it’s drinking in by endocytosis, membrane is coming from PM into cell to form vesicles

181
Q

What is the size of an inside-out vesicle compared to right-side out vesicles?

A

Inside-out vesicles are tiny compared to right-side out vesicles.

182
Q

How can we differentiate inside-out vesicles compared to right-side out vesicles?

A

Inside-out vesicles are tiny compared to right-side out vesicles, so they can separated by centrifugation.

183
Q

How are inside-out and right-side out vesicles separated in centriguation?

A

Right side spin down easily, inside out spin down harder

184
Q

What are inside-out and right-side out vesicles?

A

Right side = membranes in proper orientation

Inside out = inner membrane on outside and outer membrane inside
- Occurs due to cellular mechanisms

185
Q

Fig. 28

Label and caption this image.

A

Right-side out and inside-out vesicles

Intact cell

Leaky right-side out vesicle
Sealed right-side out vesicle

Leaky inside-out vesicle
Sealed inside-out vesicle

186
Q

Draw an image of right-side out and inside-out vesicles.

A

See Fig. 28

Right-side out and inside-out vesicles

Intact cell

Leaky right-side out vesicle
Sealed right-side out vesicle

Leaky inside-out vesicle
Sealed inside-out vesicle

(Two lamellae of the bilayer drawn in different colours)

187
Q

Additional info

Fig. 1

Explain this image.

A

N-terminal signal sequence folds into the membrane positioning positively charged resides on the cytoplasmic side, signal peptidase cleavage, nascent polypeptide extruded into ER lumen during synthesis

188
Q

Additional info

Fig. 2

Explain this image.

A

N-terminal signal sequence, signal peptidase cleavage, nascent polypeptide extruded into ER lumen during synthesis until hydrophobic stop transfer sequence synthesised, ribosome detaches from ER and completes protein synthesis in the cytoplasm

ESA 1 - Introduction to Cell Physiology and Pharmacology (10 decks)

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