Membrane Proteins Flashcards
(129 cards)
1
Q
What kinds of evidence is there for membrane proteins?
A
Functional, biochemical
2
Q
What functional evidence is there for membrane proteins?
A
Membranes have specific function, and specific function is conferred by proteins
3
Q
What specific functions do membranes have?
A
Facilitated diffusion
Ion gradients
Specificity of cell responses
4
Q
What biochemical evidence is there for membrane proteins?
A
Membrane fractionation and gel electrophoresis
Freeze fracture
5
Q
How is SDS-PAGE performed on the erythrocyte membrane?
A
Take RBC and spin to remove plasma
Put in hypertonic solution
Spin in centrifuge to give membrane pellet
Put membrane into SDS-PAGE
6
Q
What happens when you put a membrane in a hypertonic solution?
A
The cell will burst, releasing haemoglobin, leaving only the membrane
7
Q
Why does the bursting of the RBC leave only the membrane?
A
Because there are no organelles in a RBC
8
Q
What colour is a RBC membrane pellet?
A
White
9
Q
What is the white membrane pellet called?
A
Erythrocyte ghost
10
Q
What will be seen when the erythrocyte ghost is put into SDS-PAGE?
A
Seperation will give a number of bands, each corresponding to proteins found in the erythrocyte membrane
11
Q
How many major proteins are detected in the erythrocyte membrane?
A
10
12
Q
What remains in the RBC membrane after a salt wash?
A
Only band 3 and 7
13
Q
What does only bands 3 and 7 remaining after a salt wash mean?
A
That all but the proteins that produce band 3 and 7 are peripheral membrane proteins, and must be on cytoplasmic face
14
Q
Why must the proteins corresponding to bands removed by the cytoplasmic face of the membrane?
A
Since they are susceptible to proteolysis only when the cytoplasmic face of the membrane is accessible
15
Q
What do the proteins removed by the salt wash comprise?
A
The cytoplasmic skeleton
16
Q
What are proteins 3 and 7?
A
Covalently attached carbohydrate units, therefore glycoproteins
17
Q
What does the highly hydrophilic nature of the extracellular carbohydrate groups act to do?
A
Lock the orientation of the protein in the membrane by preventing flip-flop rotation
18
Q
How is the freeze fracture technique carried out?
A
Freeze cell in ice
Fracture with knife
19
Q
What happens when you fracture the frozen cell with a knife?
A
The ice crystal will break around the weakest point, which is between the two lamallae of the bilayer
20
Q
What is the result of the ice crystal breaking between the two lamallae of the bilayer?
A
The fracture pulls the two lamellae apart, taking the proteins with one of the lamellae, producing the P and C face
21
Q
What is the P face?
A
The lamellae next to cytosol
22
Q
What is the C face?
A
The lamallae next to extracellular water
23
Q
How is the freeze fracture used to visualise proteins?
A
You take the crystal, shadow at a long angle with osmium or some other electron dense metal, and so build up a ‘snow drift’ against anything sticking up, or in holes, that can then be visualised with an electron microscope
24
Q
What does the fluid mosaic theory of membrane structure say?
A
That biological membranes are composed of lipid bilayer associated with membrane proteins
25
How can the lipid bilayer be associated with membrane proteins?
May be deeply embedded in bilayer
| May be associated with surface
26
What is it called when proteins are deeply embedded in the bilayer?
Integral
27
What is called when proteins are associated with the surface?
Peripheral
28
How can proteins move in the bilayer?
Conformational change
Rotational
Lateral
29
What allows proteins to change conformation?
The fluid membrane
30
What does lateral diffusion allow?
Recruitment of a partner to perform a function
31
Can membrane proteins perform flip-flop?
No
32
Why can’t membrane proteins perform flip-flop?
It is energetically unacceptable to take a large hydrophilic protein molecule through the bilayer
Taking a big molecule such as protein through the bilayer would disrupt the structure of the bilayer, destroying ion gradients
33
How can movement of proteins in the bilayer be described?
As dynamic- happens all the time
34
What restricts protein mobility?
Aggregates
Tethering
Interactions with other cells
35
Why do aggregates restrict protein mobility?
It is more difficult to move if proteins are in aggregated structures
36
What can a protein be tethered to?
Something outside the cell
37
Can proteins move most of the time?
Yes
38
When is a membrane protein fixed?
When involved in a specific function, such as synapse or cell-cell or cell-basement membrane interactions
39
What restraints on mobility are there?
Lipid mediated effects
Membrane protein associations
Association with extra-membraneous proteins (peripheral proteins)
40
How do lipid mediated effects impact mobility?
Proteins tend to separate out into the fluid phase, or cholesterol poor regions
41
What does cholesterol determine?
How proteins may be segregating in the membrane
42
What kind of regions are more fluid?
Cholesterol poor
43
What often clusters in high cholesterol regions?
Signalling proteins
44
Why do signalling proteins often cluster in high cholesterol regions?
Because they need to stay in one specific place
45
What extra-membranous proteins can restrain mobility?
The cytoskeleton
46
What are the types of membrane proteins?
Peripheral
| Integral
47
How are peripheral membranes related to the membrane?
They are associated with the membrane- bound to the surace- but not inside
48
How are peripheral proteins bound to the surface?
By electrostatic and hydrogen bond interactions
49
How are peripheral membrane proteins removed?
By changes in pH or in ionic strength- they can be washed off by a high salt solution
50
How do integral proteins interact with the membrane?
They interact extensively with hydrophilic domains of the lipid bilayer
51
Can integral proteins be removed by manipulation of pH or ionic strength?
No
52
How are integral proteins removed?
By agents that compete for non-polar interactions, e.g. detergents and organic solvents
53
What is often true of the R groups of amino acid residues in transmembrane domains of proteins?
They are largely hydrophilic, small or polar, uncharged
54
What structure do transmembrane domains of proteins often have?
α-helical
55
How many amino acids often make up transmembrane domains?
18-22
56
What do hydropathy plots tell you?
How hydrophilic an amino sequence is
57
What can hydropathy plots be used to determine?
The transmembrane domains
58
How do hydropathy plots work?
Takes the first 20 amino acids, and gives them a score as to how many of the amino acids are hydrophilic.
It then moves down one amino acid, and takes another score
59
What would you expect the hydropathy score of a transmembrane domain to be?
Very hydrophilic
60
How are proteins always orientated?
One way or another, so specific parts of the proteins are on the inside and outside
61
Can proteins be reversed in the membrane?
No
62
What is asymmetrical orientation of proteins in biological membranes important for?
Function
63
Give an example of why asymmetrical orientation of proteins in biological membranes is important for function?
E.g. a receptor for hydrophilic extracellular messenger molecules must have a recognition site directed towards the extracellular space to function
64
How can proteins be associated in bilayers?
Peripheral protein associations
Single or multiple transmembrane domains
Post-translational lipid modification
Dolichol phosphate-linked polypeptides
65
How do peripheral protein assocations work?
Protein A is attached to protein B, which is peripherally associated with the bilayer. Protein A is therefore not directly attached to the bilayer, but protein B is
66
What happens when proteins have multiple transmembrane domains?
They have more than one part of the protein incorporated into the bilayer
67
Give two examples of posttranslational lipid modification?
Mystroylation
| Palmitoylation
68
How can post-translational lipid modification associate proteins with the bilayer?
Lipid molecules attached to the protein can anchor it into the bilayer
69
Are proteins attached by post-translational lipid modification attached solely using lipids?
They can be, or can be attached using both transmembrane domains and lipids
70
What is the result when a protein is attached using transmembrane domains and lipids?
The protein is further restricted back into the membrane, as not only is the α-helical domain locking it into the membrane, but also a fatty acid
71
What happens with dolichol phosphate-linked polypeptides?
A lipid is integrated into the bilayer. That lipid is attached to a carbohydrate via a phoshphate. The carbohydrate is attached to the polypeptide, via another phosphate on the other end
72
What is the erythrocyte cytoskeleton made up of?
A network spectrin and actin molecules
73
What kind of protein is spectrin?
Fibrous
74
What are the subunits of spectrin?
α and ß
75
Describe the structure of spectrin?
Long, floppy, rod-like molecule
76
How is the final structure of spectrin formed?
α and ß subunits wind together to form an antiparallel heterodimer
Two heterodimers form head to head associations, to form a heterotetramer of α 2 ß 2
These rods are crosslinked into networks by short actin protofilaments and band 4.1 and adducin molecules
These form interactions towards the end of the spectrin rods
77
What is the short actin protofilament made up of?
~14 actin monomers
78
What does spectrin form?
A lattice, cage like structure
79
What happens to the cage like spectrin structure?
It is grafted onto the inside of the RBC membrane
80
How is the spectrin cage attached to the membrane?
Through adapter proteins
81
How is spectrin linked to band 3 protein?
Via ankyrin
82
How is spectrin linked to glycophorin A?
Via band 4.1
83
What is the effect of attachment of integral membrane proteins to the cytoskeleton?
It restricts the lateral mobility of the membane protein
84
What is the erythrocyte cytoskeleton important for?
Maintaining deformability
85
Why is deformability necessary for erythrocytes?
So they can make their passage through capillary beds without lysis
86
Give 3 types of haemolytic anaemias
Hereditary spherocytosis
Hereditary Elliptocytosis
Ankyrin defects
87
Is hereditary spherocytosis a dominant or recessive disease?
Dominant
88
What is the defect in hereditary spherocytosis?
Spectrin is depleted by 40-50%, and so there is a reduced cage-like structure
89
What is the result of hereditary spherocytosis?
Erythrocytes round up, losing their biconcave shape, and so are less resistant to lysis
90
What does hereditary spherocytosis result in?
Anaemia
91
Why does hereditary spherocytosis result in anaemia?
Because the rounded cells get stuck in the capillary, and the plasma rushing past rips the cells apart. The cell fragments are then removed by the spleen, and so the RBC’s have a shortened in vivo survival time.
The bone marrow is unable to compensate for the reduced life span
92
What is the result of anaemia?
Reduced ability to carry oxygen, so patients are constantly tired and weak
93
What is the treatment for hereditary spherocytosis?
Blood transfusions ~every 100 days
94
What is Hereditary Elliptocytosis a defect in?
Spectrin molecules- they are present, but don’t assemble the end-to-end structure as well
95
What is a result of spectrin being less able to form their end-to-end structure in Hereditary Elliptocytosis?
They are unable to form heterotetramers, and so they form fragile, rugby ball (elliptoid) shaped RBCs
96
How are haemolytic anaemias treated?
Cytochalasin drugs
97
What do cytochalasin drugs do?
Cap the growing end of polymerising actin filaments, and so can alter the deformity of the erythrocyte
98
How are membrane proteins and those to be secreted or targeted to lysosomes synthesised?
Against the mRNA template by the ribosomes
99
What are proteins sometimes produced with?
A leader sequence of hydrophobic amino acids
100
What is the leader sequence of a protein?
18-30 amino acids with a number of basic residues at the N-terminus
101
What happens to the leader sequence as it emerges from the ribosome?
It is recognised by a signal recognition particle
102
What is a SRP?
A large protein/RNA complex
103
What does the SRP do?
Grabs the leader sequence and the ribosome and locks them together
104
What is the result of the SRP locking of the leader sequence and the ribosome?
Prevents anything from happening until it reaches the ER
105
What recognises the SRP?
A docking protein/SRP receptor
106
What happens when the SRP is recognised by a docking protein?
It brings the ribosome down onto the ER
In making the interaction with the docking protein, the SRP is released from the signal sequence of the nascent polypeptide
107
What is the result of the release of the SRP from the polypeptide?
Removes the inhibitory constraint on further translation
108
What is the leader sequence recognised by once in ER?
The signal sequence receptor (SSR) within a translocator complex (Sec61) in the ER
109
What does the SSR do?
Takes the signal and starts feeding through into lumen of ER
110
Where is the ribosome as the protein is translated?
Sitting on the ER, making the protein into the lumen, anchored to a pore complex through which the glowing polypeptide chain is extruded
111
When does protein synthesis into the ER cease?
When the C-terminal is released from the ribosome
112
What happens in the case of a secreted or lysosomal protein?
When synthesis is completed, the protein is translocated into the lumen of the ER
113
How is a protein synthesised when it needs to remain in the membrane?
As the protein is synthesised, if a stop-transfer signal is found, it sticks in the membrane
114
What is a stop-transfer signal?
A highly hydrophobic primary sequence followed directly by charged amino acids, which, in alpha-helical form, is long enough to span the hydrophobic core of the bilayer
115
What does the stop-transfer sequence from?
The transmembranous region of the protein
116
Why does the stop-transfer sequence form the transmembranous region of the protein?
Because its thermodynamically happier to be inside the hydrophobic domain of the bilayer
117
How is a membrane protein released from the protein translocator into the lipid bilayer?
A lateral gating mechanism
118
What happens as the ribosomes continues to synthesise a membrane protein after the stop-transfer signal?
It gets pushed away from the membrane, and continues to synthesise the protein in the cytoplasm
119
What is the result of the synthesis of a protein with a stop-transfer signal?
A transmembrane protein with it’s N-terminal directly into the lumen and it’s C-terminal to the cytoplasm
120
What happens to both secretory and membrane incorporated proteins?
The signal sequence if cleaved from the new protein by signal peptidases
121
When is the signal sequence cleaved from the new protein?
Even before protein synthesis is completed
122
What is likely when a protein has multiple transmembrane domains?
That the folding of the nascent protein against the constraint of the first transmembrane segment is the driving force for insertion of other domains
123
What possibly controls membrane insertion when there are multiple spanning transmembrane domains?
A series of start- and stop-transfer sequences within the primary structure
124
What assists in stabilising the partially folded growing polypeptide when there are multiple transmembrane?
The association of luminal binding proteins (e.g. BiP) related to the family of heat-shock (chaperone) proteins
125
In what manner are transmembrane domains added?
In pairs
126
How are nascent chains further processed?
As they pass from the ER and through the cis to trans Golgi
127
When does a new protein continue along the secretory pathway until?
Secretory vesicles fuse with the plasma membrane
128
What happens when secretory vesicles fuse with the plasma membrane?
Secreted proteins are released from the cell, and membrane proteins are delivered such that regions of the protein that were located in the cytoplasm during synthesis remain with this orientation
129
Why are specific carbohydrate groups on membrane proteins important?
May be important for cellular recognition to allow tissues to form an immune recognition
