Session 2.3c - Workbook Notes - Lecture 2 Flashcards
(163 cards)
1
Q
Aims:
Consider the distribution of proteins in membrane structure
A
- peripheral
- integral
generally in fluid phase (cholesterol-poor region)
2
Q
Aims:
Consider the role of proteins in membrane structure
A
Can have a role in cytoskeleton
3
Q
Aims:
Consider the importance of an asymmetric distribution of membrane proteins
A
Different distribution/orientation is due to different functions e.g. receptor for insulin must be directed extracellularly
4
Q
Aims:
Consider the importance of mechanisms for the correct insertion of membrane proteins into the lipid bilayer
A
Ensures proteins have topography
5
Q
Aims:
Consider the structure of the erythrocyte cytoskeleton.
A
Spectrin
6
Q
LO:
Outline the evidence for membrane proteins
A
Functional
Biochemical
- freeze fracture
- gel electrophoresis
7
Q
LO:
Describe how membrane proteins associate with the lipid bilayer
A
- peripheral
- integral
8
Q
LO:
Describe how membrane proteins may move
A
- conformational change
- rotational
- lateral
9
Q
LO:
Describe how membrane proteins contribute to the
cytoskeleton
A
- spectrin
10
Q
LO:
Describe how membrane proteins inserted into membranes
A
- SRP/SS/DP/SP
11
Q
LO:
Discuss how the correct orientation of membrane proteins
achieved
A
- SRP/SS/DP/SP
- SP cleavage - N-terminal facing lumen
12
Q
What is the current model of membrane structure?
A
The lipid mosaic theory (Singer-Nicholson model)
13
Q
What is the Singer-Nicholson model?
A
The lipid mosaic theory of membrane structure
14
Q
What are biological membranes composed of?
A
A lipid bilayer with associated membrane proteins, which may be integral or peripheral.
15
Q
What is an integral membrane protein?
A
Proteins that are deeply embedded into the bilayer
16
Q
What is a peripheral membrane protein?
A
Proteins that are associated with the surface
17
Q
What are deeply embedded bilayer proteins called?
A
Integral membrane proteins
18
Q
What are proteins associated with the surface called?
A
Peripheral membrane proteins
19
Q
Fig. 5
Caption and label this image.
A
Extracytoplasmic Surface
Cytoplasmic surface
Hydrophilic
Hydrophobic
20
Q
Draw the Singer-Nicholson model.
A
See Fig. 5
Extracytoplasmic Surface
Cytoplasmic surface
Hydrophilic
Hydrophobic
(Hydrophobic regions fully enclosed
Hydrophilic areas exposed to the outside/water)
21
Q
Describe peripheral membrane proteins.
A
- Deeply embedded in the bilayer
- Bound to the surface of membranes by electrostatic and hydrogen bond interactions
- Can be removed by changes in pH or ionic strength
22
Q
How are peripheral membrane proteins bound to the surface of membranes?
A
By electrostatic and hydrogen bond interactions.
23
Q
How can we remove peripheral proteins from the membrane?
A
By changes in pH or ionic strength
24
Q
Which type of protein is bound by electrostatic and hydrogen bond interactions?
A
Peripheral
25
Which type of protein can be removed from the membrane by changes in pH or ionic strength?
Peripheral only (not integral)
26
What do integral membrane proteins interact with?
Extensively with the hydrophobic regions of the lipid bilayer
27
Which proteins interact extensively with the hydrophobic regions of the lipid bilayer?
Integral (they pass through the membrane)
28
What can integral membrane proteins not be removed by?
Manipulation of pH or ionic strength
29
What do integral membrane proteins require for removal?
Agents which compete for the non-polar interactions in the bilayer.
30
Give two examples of agents which can remove integral membrane proteins.
- Detergents
| - Organic solvents
31
Explain how integral membrane proteins are removed and how? (3 marks)
They cannot be removed by manipulation of pH or ionic strength (1 mark) because the bonds are too strong.
They therefore require agents which compete for the non-polar interactions in the bilayer (1 mark).
These include detergents and organic solvents (1 mark).
32
What is asymmetrical orientation of membrane proteins?
Certain proteins must have an orientation, e.g. receptors may need to face extracellularly rather than intracellularly for function.
33
Why do we need asymmetrical orientation of proteins?
Important for function
34
Give an example of a need for asymmetrical orientation in membrane proteins.
A receptor for a hydrophilic extracellular messenger molecule, such as insulin, must have its recognition site directed towards the extracellular space to be able to function.
35
What sort of molecule is insulin?
A hydrophilic extracellular messenger molecule
36
Why must the insulin receptor face extracellularly?
Because insulin is a hydrophilic extracellular messenger molecule, so needs to face that way for function.
37
Why do we look at the erythrocyte membrane?
It is a model plasma membrane
38
What do we use as a model plasma membrane?
The erythrocyte membrane (because it has no organelles so is the simplest membrane).
39
How can we prepare erythrocyte ghosts?
By osmotic haemolysis to release cytoplasmic components
40
What does osmotic haemolysis of erythrocyte membranes do?
Release its cytoplasmic components.
41
How can we analyse ghost membranes?
By gel electrophoresis
42
What have we discovered in analysis of ghost membranes?
Over 10 major proteins
43
What are the major erythrocyte membrane proteins found by gel electrophoresis called?
```
The major ones have been numbered
1
2
3
4.1
4.2
5
6
7
etc
```
44
Most of the membrane proteins in erythrocyte membranes are peripheral proteins. How do we know?
Most of these proteins are released when ghost membranes are treated with high ionic strength medium or by changing the pH
45
Most of the membrane proteins in erythrocyte membranes are released when treated with high ionic strength
medium or by changing the pH. What does this indicate?
Most of the major proteins are peripheral proteins.
46
How do we know that most of the erythocyte proteins are located on the cytoplasmic face?
They are susceptible to proteolysis only when the cytoplasmic face of the membrane is accessible.
47
Many erythrocyte membrane proteins are susceptible to proteolysis only when the cytoplasmic face of the membrane is accessible. What can we infer from this?
These peripheral proteins must be located on the cytoplasmic face
48
What are 2 major integral membrane proteins of erythrocytes?
Protein bands 3 and 7
49
What do protein bands 3 and 7 in erythrocyte membranes have in common? Give TWO answers
- They are both integral proteins
| - They are both glycoproteins
50
Protein bands 3 and 7 can only be dissociated from the red cell membrane by detergents. What does this suggest?
They are integral proteins.
51
How do we know protein bands 3 and 7 in the RBC membrane are integral proteins?
They can only be dissociated from the red cell membrane by detergents
52
Protein bands 3 and 7 of the RBC membrane contain covalently attached carbohydrate units. What term do we use to describe these molecules?
Glycoproteins.
53
Proteins bands 3 and 7 are glycoproteins. Describe their structure.
Both proteins contain covalently attached carbohydrate units
54
How are the carbohydrate units attached in glycoproteins?
Covalently
55
What is the nature of the carbohydrate groups in glycoproteins?
They are highly hydrophilic and extracellular
56
Which part of the glycoprotein is hydrophilic?
The carbohydrate group
57
Which part of protein bands 3 and 7 are extracellular?
The hydrophilic carbohydrate group (because they are glycoproteins).
58
What is the significance of protein bands 3 and 7 being glycoproteins?
The highly hydrophilic nature of the extracellular carbohydrate groups acts to lock the orientation of the protein in the membrane by preventing flip-flop rotation
59
How is flip-flop rotation prevented in glycoproteins?
The highly hydrophilic nature of the extracellular carbohydrate groups acts to lock the orientation of the protein in the membrane by preventing flip-flop rotation
60
What carbohydrate structures are available on different membrane proteins?
A great variety of carbohydrate structures is possible on different proteins.
61
What is the significance of having a great variety of carbohydrate structures on membrane proteins?
Specific carbohydrate groups on membrane proteins may be important for cellular recognition to allow tissues to form and in immune recognition.
62
Give 2 functions of the carbohydrate group on membrane proteins
- Cellular recognition to allow tissues to form
| - Immune recognition.
63
What is the cytoskeleton?
The membrane skeleton
64
What is the term used to describe the membrane skeleton?
The cytoskeleton
65
What type of proteins are the cytoskeleton proteins?
Peripheral membrane proteins
66
Give 2 important peripheral membrane proteins of the erythrocyte membrane.
Spectrin
Actin
- part of the cytoskeleton
67
What is the erythrocyte cytoskeleton made up of?
A network of spectrin and actin molecules
68
What do spectrin and actin molecules compose?
The erythrocyte cytoskeleton
69
Describe the structure of spectrin
- long, floppy rod-like molecule
- a and b subunits wind together to form an antiparallel
heterodimer
- two heterodimers then form a head-to-head association
to form a heterotetramer of a2b2.
70
What does spectrin look like under an electron microscope?
A long, floppy rod-like molecule
71
What subunits does spectrin have?
Alpha and beta
72
How are the two subunits of spectrin interrelated?
a and b subunits wind together to form an antiparallel
| heterodimer
73
How are molecules of spectrin interrelated?
```
two heterodimers (of a and b subunits) then form a head-to-head association
to form a heterotetramer of a2b2.
```
74
What is the 3D structure of spectrin?
- a and b subunits form an antiparallel heterodimer
| - two heterodimers bt head-to-head association form a heterotetramer of a2b2
75
How are the spectrin rods crosslinked into networks?
By short actin protofilaments (~14 actin monomers)
76
Describe the role of short actin protofilaments in spectrin.
Allows spectrin rods to be crosslinked into networks.
77
What are short actin protofilaments made up of?
~14 actin monomers
78
~14 actin monomers are known as?
Short actin protofilaments
79
What do Band 4.1 and Adducin do?
Form interactions towards the ends of the spectrin rods
80
What forms interactions towards the ends of the spectrin rods?
Band 4.1 and Adducin
81
What is the role of Band 4.1?
Forms interactions towards the ends of the spectrin rods, alongside Adducin
82
What is the role of Adducin?
Forms interactions towards the ends of the spectrin rods, alongside Band 4.1
83
How is the spectrin-actin network attached to the membrane?
Through adapter proteins
84
What do adapter proteins do?
Attach the spectrin-actin network to the membrane
85
What is ankyrin also known as?
Band 4.9
86
What is Band 4.9 also known as?
Ankyrin
87
What is the role of ankyrin?
(nkyrin/Band 4.9) Links spectrin and Band 3 protein (found in the membrane)
88
What is the role of band 4.9?
(Ankyrin/Band 4.9) Links spectrin and Band 3 protein (found in the membrane)
89
What is the role of band 4.1?
Links spectrin and glycophorin A (found in the membrane)
90
Name 2 adaptor proteins
Ankyrin (band 4.9)
| Band 4.1
91
What is the significance of attaching integral membrane proteins to the cytoskeleton?
Restricts lateral mobility of the membrane protein
92
How is lateral mobility of membrane protein restricted?
By attaching integral membrane proteins to the cytoskeleton
93
Fig. 6
Label the image
```
Band 3
Glycophorin A
Membrane
Ankyrin (band 4.9)
Band 4.1
a b Spectrin
Adducin
Band 4.1
Actin
```
94
Draw and label the erythrocyte cytoskeleton.
See Fig. 6
```
Band 3 } TM protein
Glycophorin A } TM protein
Membrane
Ankyrin (band 4.9) }
Band 4.1 } adaptor protein
a b Spectrin
Adducin
Band 4.1
Actin
```
95
What is the clinical significance of the erythrocyte cytoskeleton?
The erythrocyte cytoskeleton is a very important structure in maintaining the deformability necessary for erythrocytes to make their passage through capillary beds without lysis
96
What is erythrocyte deformability?
The ability of erythrocytes (red blood cells, RBC) to change shape under a given level of applied stress, without hemolysing (rupturing)
97
What allows erythrocyte deformability?
(Deformability - the ability to change shape under pressure, e.g. through tiny capillaries, without lysis)
The cytoskeleton
98
What genetic pattern does Hereditary Spherocytosis follow?
Dominant
99
What is the incidence of hereditary spherocytosis?
1:2000
100
What is the pathophysiology for the most common form of hereditary spherocytosis?
Spectrin levels are depleted by 40-50%
101
How much are spectrin levels depleted by in hereditary spherocytosis?
40-50%
102
Spectrin levels are depleted by 40-50% in what disease?
Hereditary spherocytosis
103
What happens to the cells in hereditary spherocytosis?
The cells round up and become much less resistant to lysis during passage through the capillaries
104
What is the most common form of haemolytic anaemia?
Hereditary spherocytosis
105
What clears the cells in hereditary spherocytosis?
The spleen
106
How can hereditary spherocytosis lead to haemolytic anaemia?
The shortened in vivo survival of red blood cells and the inability of the bone marrow to compensate for their reduced life span lead to haemolytic anaemia
107
State 2 causes of haemolytic anaemia from hereditary spherocytosis.
- The shortened in vivo survival of red blood cells
| - The inability of the bone marrow to compensate for their reduced life span
108
What other forms of hereditary spherocytosis exist?
Where mutated cytoskeletal elements with dysfunctional binding sites for
other components are expressed
109
Give 9 facts about hereditary spherocytosis.
The most common form:
- DOMINANT
- INCIDENCE ~1:2000
- SPECTRIN levels DEPLETED by 40-50%
- Cells ROUND up
- Cells become MUCH LESS resistant to lysis in capillaries
- CLEARED by the SPLEEN
- SHORTENED in vivo SURVIVAL of RBCs AND
- INABILITY of BONE MARROW to COMPENSATE for reduced lifespan leads to HAEMOLYTIC ANAEMIA
- Other forms exist where MUTATED CYTOSKELETAL ELEMENTS with DYSFUNCTIONAL BINDING SITES for OTHER COMPONENTS are expressed
110
What is the most common defect in hereditary elliptocytosis?
Spectrin is unable to form heterotetramers
111
What disease results from a spectrin molecule being unable to form heterotetramers?
Hereditary elliptocytosis
112
What is the pathophysiology of hereditary elliptocytosis?
Spectrin is unable to form heterotetramers resulting in fragile elliptoid cells.
113
What is the shape of the cells in heredtiary elliptocytosis?
Elliptoid
114
What are the features of hereditary elliptocytosis?
- Spectirn molecule unable to form heterotetramers
| - Fragile elliptoid cells
115
What is the difference in pathophysiology of hereditary spherocytosis and hereditary elliptocytosis?
Hereditary spherocytosis: not as much spectrin (or other cytoskeletal component) being produced so WEAK cytoskeleton, resulting in SPHERICAL cells
Hereditary elliptocytosis: spectrin being produced but cannot form heterotetramers (a2b2) resulting in FRAGILE cytoskeleton and ELLIPTOID cells
116
What is the mechanism of action of cytochalasin drugs?
Cap the growing end of polymerising actin filaments
117
What drugs cap the growing end of polymerising actin filaments?
Cytochalasin drugs
118
Why do we not use cytochalasin drugs in person?
They cap the growing end of polymerising actin filaments, so can alter the deformability (ability to squeeze through small places without lysis) of the erythrocyte.
119
What can affect the erythrocyte cytoskeleton?
- Hereditary spherocytosis
- Hereditary elliptocytosis
- Cytochalasin drugs
120
Why are we concerned about the effect on the erythrocyte cytoskeleton?
Bc affecting it can lead to reduced deformability of RBCs, leading to lysis and therefore haemolytic anaemia.
121
What is similar in cytosolic proteins and membrane protein synthesis?
Like cytosolic proteins, membrane proteins (and those to be secreted or targeted to lysosomes) are synthesised against the messenger RNA template by ribosomes
122
Which proteins are initially synthesised in the cytosol but then prevented from further synthesis?
- Membrane proteins
| - Those to be secreted or targeted to lysosomes (secreted proteins) e.g. insulin
123
What happens to a membrane or secreted protein synthesis in the cytosol.
Before synthesis progresses very far the translation of these proteins is halted until the ribosome has been transferred to the rough ER
124
What is the signal sequence made up of?
A characteristic hydrophobic amino acid sequence of 18 - 30 amino acids with a number of basic residues at the N-terminus of the nascent polypeptide
125
Name 4 properties of the signal sequence.
- Hydrophobic AA sequence
- 18-30 AAs long
- Number of basic residues included
- At the N-terminus
126
What is the signal or leader sequence?
A characteristic hydrophobic amino acid sequence of 18 - 30 amino acids with a number of basic residues at the N-terminus of the nascent polypeptide which, upon recognition by SRP, arrests further protein synthesis in the cytoplasm.
127
What is the signal sequence recognised by?
The signal recognition particle
128
What is the signal recognition particle (SRP)?
A large protein/RNA complex which recognises the signal sequence.
129
What is the function of SRP?
Binding of the SRP to the growing polypeptide chain and the ribosome locks the ribosome complex and prevents further protein synthesis while the ribosome is in the cytoplasm.
130
How does SRP prevent further protein synthesis when the ribosome is in the cytoplasm?
By recognising the signal sequence and locking the growing polypeptide chain and the ribosome in place with itself.
131
What prevents further synthesis of protein whilst a membrane/secreted protein is in the cytoplasm?
Recognition of the signal sequence by SRP, binding the ribosome and growing nascent polypeptide in place in the cytoplasm.
132
Where do membrane/secreted proteins need to be directed to?
The membrane/RER membrane respectively
133
What is SRP recognised by?
An SRP RECEPTOR or DOCKING PROTEIN
134
What does an SRP receptor/docking protein recognise?
The signal recognition particle (SRP)
135
Where is the SRP receptor/docking protein found?
On the ER membrane.
136
What happens when the SRP and docking protein interact?
In making the interaction with the docking protein, the SRP is released from the signal sequence of the nascent polypeptide removing the inhibitory constraint on further translation
137
How does translation of the polypeptide re-continue after arrest from SRP? (Membrane/secreted protein biosynthesis)
SRP binds to the docking protein, releasing itself from the signal sequence and therefore releasing its inhibition.
138
What does the signal sequence interact with after being released from SRP?
The signal sequence then interacts with a signal sequence receptor (SSR) within a protein translocator complex (Sec61) in the ER membrane, which directs further synthesis through the ER membrane
139
How does further synthesis of the proitein continue after the signal sequence is released?
The signal sequence then interacts with a signal sequence receptor (SSR) within a protein translocator complex (Sec61) in the ER membrane, which directs further synthesis through the ER membrane
140
Where is the signal sequence receptor?
Within a protein translocator complex (Sec61) in the ER membrane
141
How is further synthesis of the protein directed through the ER membrane after release of SRP?
The signal sequence then interacts with a signal sequence receptor (SSR) within a protein translocator complex (Sec61) in the ER membrane, which directs further synthesis through the ER membrane
142
What causes the protein to be synthesised through the membrane?
The ribosome becomes anchored to this pore complex, through which the growing polypeptide chain is extruded
143
What happens to a secreted or lysosomal protein after the ribosome is anchored to the pore complex?
In the case of a secreted or lysosomal protein, synthesis is completed and the nascent protein is translocated into the lumen of the ER
144
What happens to a membrane protein after the ribosome is anchored to the pore complex?
For membrane proteins the passage of the protein through the membrane must be arrested.
145
What's the difference between membrane protein synthesis and secreted protein synthesis?
In the case of a secreted or lysosomal protein, synthesis is completed and the nascent protein is translocated into the lumen of the ER.
For membrane proteins the passage of the protein through the membrane must be arrested.
146
How are membrane proteins arrested in the membrane?
Via a stop transfer signal
147
What is the stop transfer signal?
A region of highly hydrophobic primary sequence in the growing polypeptide of between 18 and 22 amino acids long
148
What follows the 'stop' part of the stop transfer signal?
Charged amino acids which, in a-helical form, is long enough to span the hydrophobic core of the bilayer
149
What does a region of 18-22 highly hydrophobic AAs in the growing polypeptide suggest?
A stop transfer signal
150
What directly follows the stop transfer signal?
Charged amino acids
151
What is the relevance of the stop transfer signal?
This sequence
| forms the trans-membranous region of the protein
152
What releases the membrane protein from the protein translocator into the lipid bilayer?
A lateral gating mechanism
153
What does the lateral gating mechanism in membrane protein synthesis do?
Releases the membrane protein from the protein translocator into the lipid bilayer.
154
What is the last step of membrane protein synthesis after release of the stop transfer signal?
The ribosome then presumably detaches from the ER and protein biosynthesis continues in the cytoplasm
155
How do we get a protein with N-terminal directed to the lumen and C-terminal to the cytoplasm?
- Normal secreted protein synthesis pathway
- Once bound to the ER, passage of the protein through membrane must be arrested
- Stop transfer signal is recognised which can span the membane
- Lateral gating mechanism releases membrane protein from protein translocator
- Ribosome detaches from ER and continues in the cytoplasm
156
Using the most basic method of membrane protein synthesis, what is the orientation of membrane proteins?
N-terminal - lumen
C-terminal - cytoplasm
(Self note: C for cytoplasm)
(*Only need to know this method, do not need to know further*)
157
What happens to the signal sequence in membrane/secretory proteins?
It is cleaved by signal peptidase
158
What is the function fo signal peptidase?
To cleave the signal sequence
159
When does signal peptidase act?
Before protein synthesis is completed
160
Fig. 8 (left)
Label and caption the image
Secretory Protein Biosynthesis
5' - 3'
- SRP
- Docking protein
- Signal sequence
- Signal sequence receptor / protein translocator complex (Sec61)
- Signal Peptidase
161
Draw the pathway for secretory protein biosynthesis.
See Fig. 8 (left)
Secretory Protein Biosynthesis
5' - 3'
- SRP
- Docking protein
- Signal sequence
- Signal sequence receptor / protein translocator complex (Sec61)
- Signal Peptidase
162
Fig. 8 (right)
Caption and label the image
Membrane Protein Biosynthesis
Cytoplasm
3'
ER lumen
Stop transfer signal
- Signal sequence
- Signal sequence receptor / protein translocator complex (Sec61)
- Signal Peptidase
163
Draw the pathway for membrane protein biosynthesis.
See Fig. 8 (right)
Membrane Protein Biosynthesis
Cytoplasm
3'
ER lumen
Stop transfer signal
- Signal sequence
- Signal sequence receptor / protein translocator complex (Sec61)
- Signal Peptidase
