Solute and protein transport Flashcards
(235 cards)
1
Q
What does the ABC stand for in ABC systems?
A
ATP Binding Casette.
2
Q
ABC systems are commonly found in bacteria and archaea. True or false?
A
False, they are also commonly found in euks and are one o the largest families of transporters.
3
Q
How may times can ABD systems be found in one organism?
A
100’s.
4
Q
ABC systems are broad systems found multiple times in the genome. True or false?
A
False. They can sometimes be specific systems.
5
Q
ABC systems are very versatile. What can they transport (5 things)?
A
- Amino acids
- Organic acids.
- Sugars.
- Vitamins.
- Inorganic ions.
6
Q
What can the substrate affinity of ABC systems be described as?
A
High.
7
Q
What does the high affinity that ABC systems have to their substrates make them ideal for?
A
Savaging nutirents.
8
Q
What two types of processes can ABC systems be involved in?
A
Efflux or uptake.
9
Q
ATP can drive efflux in ABC systems. What is this useful for?
A
Excretion of solutes, antibiotics and toxic compounds.
10
Q
How does the arrangement of the protein subunits of the ABC system vary between bacteria and eukaryotes?
A
In eukaryotes the proteins are separate and in bacteria they are fused in into a single polypeptide with distinct domains.
11
Q
ABC systems have a great medical importance. Give two example of this.
A
In humans CF is caused by a mutation in a ABC type Cl- transporter CFTR.
Multi drug resistant tumours have a MDR system that can expel drugs.
12
Q
How many subunits are ABC transporters made up from in bacteria?
A
3-5.
13
Q
What subunit of ABC transporters has the ATPase activity?
A
ABC.
14
Q
ABC subunits allow for high accumulation ratios of ____.
A
10^5.
15
Q
What do bacterial uptake ABC proteins depend on?
A
Soluble periplasmic solute binding protein.
16
Q
Both +ve and -ve bacteria need soluble periplasmic solute binding proteins for ABC transporters to work. How is this possible in +ve bacteria, which have no periplasm?
A
They can anchor to the cell membrane.
17
Q
What are ABC transport systems sensitive to?
A
Osmotic shock.
18
Q
What Km values correlate to a high substrate affinity?
A
Low.
19
Q
Gram negative bacteria allow for a good diagnostic test to determine whether a particular transporter depends on a periplasmic binding protein. Explain this test in 5 steps.
A
- Cell is treated with 0.5M sucrose and 1mM EDTA.
- EDTA acts as a cheating agent to bind Mg2+ and Ca2+ ions. These are important for the stability of the outermsmbrane meaning the outer membrane becomes permeable.
- Plasmolysis occurs (H2O leaves.)
- Cells can be diluted with large volumes of H2O due to the leaky membrane. The concentration of periplasmic binding proteins is now lower due to the leaky membrane but INTACT cell wall.
- See if solute transport still occurs.
20
Q
Do efflux systems from ABC transporters need periplasmic binding proteins?
A
No.
21
Q
What three subunits make up the CFTR transporter?
A
D, R and E. R is the regulatory domain.
22
Q
What two subunits make up the MDR transporter?
A
D and E.
23
Q
How many domains do periplasmic binding proteins have?
A
2, with a cleft in between.
24
Q
How many conformations of periplasmic binding proteins are there?
A
2.
25
What are the two conformations of periplasmic binding proteins?
BP- open
BP-L closed
There is a large conformational change between these.
26
Why do periplasmic binding proteins have a high substrate affinity?
Kon is much faster than Koff.
27
How is the ligand maintained in the periplasmic binding protein?
Mainly H bonds.
28
What periplasmic binding protein conformation interaction with the membrane protein?
BP-L.
29
District families of BP exist. What do these have in common?
Related in sequence and the substrate they bind.
30
How many transmembrane helices are integral membrane proteins usually made from?
6.
31
How are integral membrane proteins arranged?
Fold to form a transporter modular system these can interact with the periplasmic binding protein. Uses energy provided by ATP hydrolysis from the ABC protein.
32
What are the key motif's of the ABC protein complexs that are involved in binding of ATP?
Walker A and Walker B.
33
What is the sequence for Walker A?
GXXXXGK(S/T)
34
What is the sequence for Walker B?
hhhhDE (h any hydrophobic residues)
35
What part of Walker A is essential in nucleotide binding?
K.
36
What is the role of D in Walker B?
Coordinates Mg2+.
37
What is the role of E in Walker A?
ATP hydrolysis.
38
How much ATP is hydrolysed by the ABC transporter per mole of solute transported?
2.
39
What do ABC transporters form a related family with?
ATP binding proteins involved in solute transport in cell division and protein export.
40
Why is the mechanism of solute binding to the periplasmic protein well understood?
As the proteins are easily crystallised.
41
Why is the transporter mechanism of the ABC transported harder to determine?
As it is difficult to crystallise membrane proteins and ABC protein interactions are difficult to maintain.
42
What ABC transporter has had its structure determined?
Vitamin B12 transporter in E.coli (BtuCDF).
43
What are the three types of solute translocating ATPases?
F, V, P.
44
What type of solute translocating ATPase is revisable and includes ATP synthase?
F.
45
Where is the V type ATPase only found?
Eukaryotic vaculor membrane.
46
What are the key points regarding the P type ATPase?
Unidirectional and form a phosphorylated intermediate during transport.
47
What do P type Atlases often transport?
Metal ions.
48
Name one example of a prokaryotic P type ATPase.
Kdp K+ uptake system.
49
What are the 4 main components of the Kdp K+ uptake system and what are their roles?
1. KdpA - Potassium transport subunit.
2. KdpB - ATPase
3. KdpC - Accessory pigment
4. KdpF - Accessory pigment
50
What provides the energy for decarboxylation driven transporters?
The release of free energy from the decarboxylation of certain organic acids.
51
What solute is normally involved in decarboxylation driven transport?
Na+.
52
What is an example of a decarboxylation transporter?
Oxaloacetate decarboxyalse.
53
What can oxaloacetate be converted into carbon dioxide and _____.
Pyruvate and CO2.
54
How many subunits is oxaloacetate decarboxylase made up of?
3. (alpha, beta and gamma)
55
What happens in the alpha subunit of oxaloacetate decarboxylase?
Carboxyl transport to biotin molecule bound to lysine.
56
What happens in the beta subunit of oxaloacetate decarboxylase?
Decarboxylation is coupled to Na+ transport.
57
What is the purpose of the gamma subunit of oxaloacetate decarboxylase?
Stabilises the enzyme.
58
What can oxaloacetate decarboxylase be described as?
An electrogenic pump that separates a Na+ motive force across the membrane.
59
What is the definition of a secondary transport system?
A transport system that uses the free-energy in the pre-existing electrochemical gradient across the membrane to move a solute against its concentration gradient.
60
What are the four types of secondary transporters?W
1. Uniport.
2. Symport.
3. Antiport.
4. TRAP.
61
What can only be transported through uniport transporters?
Postive ions.
62
Why are uriporters not that common?
As they are restricted and not important to bacterial physiology.
63
What do symport transporters do?
Move a solute through the membrane by coupling it to the movement if a coupling ion (usually H+ or Na+) which moves down its concentration gradient. The solute does not need to be positively charged. Both move in the same direction
64
Why can uniport systems only move positive ions?
As only positive ions can be drawn in as the membrane potential is negative.
65
How do antiport transporters work?
They move two solutes in opposite directions, relying on the concentration gradient of solute 2. If these molecules are not charged they do not involve the pmf force.
66
What are 5 common properties of symport, uniport and antiport transporters?
1. Made of one gene product (single protein)
2. Distance evolutionary relationship between them with many being part of the MFS superfamily (bac and euks.)
3. Reversible and can catalyse facilitated diffusion in absence of electrochemical gradient.
4. Lower affinity than ABC systems (so higher Vmax values).
5. Insenstive to osmotic shock as no peripalsmic binding proteins.
67
Why is it easier to study secondary transport systems than primary transport systems?
As they can be studied as membrane proteins as most only consist of one protein.
68
How are would you make a membrane vesicle prep to study secondary transport systems?
Use a french press and a low spin speed to make a cell free extract from intact cells. This is then spun at a high speed allowing the membranes to spontaneously circulise.
69
What are three advantages of using membrane studies over intact cells?
1. Can study transport in the absence of metabolism.
2. Can easily study the relationship between transport activity and membrane energisation when an electron donor (such as NADH) is added to electron transport.
3. Membrane vesicles are much more sensitive to inhibitors such as ionophores. This makes it easier to collapse ion gradients and study energy coupling.
70
What is the best studied symport system biochemical and energetically?
LacY permase.
71
Besides ABC systems what are the most common solute transport systems?
Symport systems.
72
What experiment, carried out by West and Mitchell in 1972, allowed symprot systems to be studied?
Lactose was added to intact E.coli cells. The external pH was measured with a high sensitivity pH electrode. Proton symport with lactose caused alkalisation of the medium. When an uncoupler was added this does not happen.
73
When a -ve molecule such as lactate is moved via symprot the driving force is only determined by the deltapH, which is too small. How does the cell overcome this?
2 H+ ions are moved.
74
When was the lac permase (LacY) structure determined and who determined it?
2003 by Kaback.
75
How many transmembrane helices does LacY consist of?
12.
76
What are the the conformations of LacY?
Inwards and outwards.
77
What proceeds substrate binding in LacY?
Protonation.
78
What can be moved via antiport transporters?
Solute and carrier ion or two solutes.
79
What are antiport transporter's often involved in?
Metabolic pathways to exchange precursors for product.
80
What is an example of an antiport system?
Fumerate/ succinate exchange via the DcuB protein.
81
What can the fumerate/ succinate exchange system be described as?
Electroneutral. It saves energy in aerobic conditions.
82
What are the two main parts of the fumigate / succinate exchange system and what are they for?
DcuB protein which imports fumigate into the cytoplasm via the exportation of succinate (product).
Frd protein which is the active site (cytoplasm side). This converts fumerate to succinate via the addition of two electrons and two hydrogens.
83
What is the rate of the fumerate/ succinate exchange system (antiporter)?
High rate.
84
Some antiporters are electrogenic. What does this mean?
They generate charge across the membrane.
85
Hoe can electrogenic transporters generate charge?
They can transport solutes that have a different charge.
86
What can some anaerobic bacteria use to generate a pmf in absence of an electron transport chain?
Electrogenic transporters.
87
Name one organism that does not contain cytochromes resulting in it using an electrogenic transporter to generate a pmf.
Oxalobacter formigenes.
88
What transporter does Oxalobacter use to generate a pmf and how does it do this?
OxIT transporter.
Oxalate (-2) transported into the cell. This is converted into formate (-1) via oxalate decarboxylase. Formate is then exported out of the cell.
89
What enzyme is involved in the fumerate/ succinate exchange system?
Fumigate reductase.
90
What produces oxalate?
Plants.
91
The 3D structure has been obtained for the GIpT protein. What did this show?
the electronetural exchange of inorganic phosphate for glycerol-3-phosphate. The outwardly directed phosphate gradient drives the uptake of G3P which can be used as a carbon source. This is similar to how Lac permase works and shows the structural/ function relationship in antiportes.
92
What is the full name of TRAP transporters?
Tripartite ATP Independent Periplasmic transporters.
93
What drives TRAP transporters?
Pmf.
94
What is usually about TRAP transporters?
They have a high affinity to solutes and use periplasmic binding proteins, unlike other secondary transporters.
95
What photosynthetic bacterium were TRAP transporters discovered in in Kelly/s lab in the 1990's?
Rhodobacter caspulatus.
96
What do the photosynthetic bacterium Rhodobacter caspulatus use TRAP transporters for?
As an uptake system for C4 dicarboxylates such as malate, succinate and fumerate.
97
What genes encode for the TRAP transporter found in Rhodobacter capsulates?
dctPQM genes.
98
What does the DctP gene encode?
Periplamic binding proteins (these evolved independently to the periplasmic binding proteins found in ABC transporters.)
99
What does the DctQ gene encode?
4 TM helices which currently have an unknown function.
100
What does the DctM gene encode?
12 TM helices which make up the transport module.
101
How is it known that the periplasmic binding proteins found in TRAP systems evolved separately to the ones found in ABC systems?
They have similar structures but different sequences.
102
Is there a structure/ function relationship between anti porters?
Yes. Lac premase has a similar mechanism to TRAP.
103
Where have TRAP transporters now also been found?
In common bacteria and archaea. NOT in euks.
104
What do all TRAP transporters seem to contain?
A carboxylate group.
105
What is the function of TRAP transporters thought to be?
To provide a high affinity uptake system for specialised compounds which have no ATP systems.
106
What substrates can be taken up by TRAP transporters (5)?
1. C4 dicarboxylic acids.
2. Sialic acids.
3. Glutamate.
4. Pyruvate.
5. 4- chlorobenzoate.
107
Why does the hydrophobic lipid barrier any as towards charged hydrophilic molecules?
A diffusion barrier.
108
What are two examples of polar uncharged molecules can diffuse trout the hydrophobic lipid membrane?
Long chain fatty acids and gases.
109
Why can uncharged non polar substrates diffuse through the bilayer?
As they have a high enough diffusion energy to support growth.
110
How can transport into the membrane be measured (8 steps) ?
1. Radiolabel solute of interest with C14 and H3.
2. Incubate with physiological buffer at optimum pH and growth temperature in the presence of oxygen.
3. When oxygen is added add the radiolaballed substrate.
4. Take samples at frequent time intervals ( 10-20 seconds.)
5. Filter cells rapidly onto nitrocellulose filters using a vacuum pump.
6. Add the filters and the cells to a vial of scintillation fluid which contains a chemical that emits light.
7 Beta particles from C14 detected and counted on a scintillation counter.
8. Rate determined ( disintegrations per minute over unit time.)
111
Why is it important that oxygen is included when measuring transport.
As it allows electron transport and the subsequent generation of ATP.
112
What are the three main characteristics of simple diffusion?
1. Non saturatable.
2. Not inhibited.
3. Energy in the solute gradient drives transport.
113
What two processes are involved in carrier mediated transport?
1. Facilitated diffusion.
| 2. Active transport.
114
Why do saturation kinetics apply to carrier mediated transport?
As it relies on a solute binding to a carrier.
115
What is inhibited by energy coupling inhibitors?
Facilitated diffusion and active transport.
116
What do uncouplers do?
Collapse the PMF.
117
What are two examples of uncouplers?
CCP and FCCP.
118
What do ionophores do?
Dissipates membrane potential.
119
What are the four types of energy transduction inhibitors?
1. Uncouplers.
2. Ionophores.
3. ATP synthase inhibitors.
4. ATP hydrolysis inhibitors.
120
What is an example of an ionophore?
Valinomycin.
121
What is DDCD^2 an example of?
ATP synthase inhibitor.
122
What is the accumulation ratio?
Concentration of solute inside cell / concentration of solute outside cell.
123
What is the maximum AR for diffusion?
1.
124
What AR values can active transport have?
10^2 - 10^5.
125
Different solute transporters have different _____.
AR values.
126
What do you need to use to measure AR values and why?
Non metabolisable analogues off natural substrates as these get metabolised. This includes fluroglucose.
127
What is primary active transport?
Active transport that depends on the direct hydrolysis of chemical bonds to release free energy. ATP is most commonly used but in theory any compound with a high free energy of hydrolysis can be used, including phosphoenol pyruvate and oxaloacetate.
128
What is secondary active transport?
Active transport than depends on a pre-existing ion gradient across the cytoplasmic membrane. This is often the PMF or one of its components (membrane potential or pH difference.) In principle this can be driven my any chemiosmotic ion gradient, including Na+ which is used in some marine bacteria.
129
What can channels be described as?
Multimeric pores.
130
Although channels can transport a wide variety of solutes what normally travels through them?
Hydrophilic solutes via diffusion.
131
Channels can be gated in response to what?
Membrane potential.
132
Why is it important that some channels are gated and controlled?
As if not controlled can lead to the collapse of the pmf and cell death.
133
Where are porins found?
The outermembrane of bacteria.
134
What membrane in bacteria has the pmf?
Inner.
135
What do porins allow?
Entry of molecules into the periplasm.
136
Although porins are non specific how are some porins selective?
Through size. Solutes up to 600 da can pass.
137
What is LamB?
Maltose porin in E.coli.
138
What is the structure of porins?
B barrel basket structure.
139
What assembles porins on the outer membrane?
A dedicated set of periplasmic and outermembrane folding proteins.
140
How do gated porins open?
Energy from the PMF in the inner membrane is transducer through the ExbB-ExbD-TonB complex to open the pore.
141
How does TonB gate the FhuA pore?
PMF driven movement.
142
What is the Fhu porin?
An iron sidrephore (ferrichrome) that captures iron for bacterial cell growth.
143
What organisms contain the phosphotransferase system?
Gram +ve and -ve bacteria.
144
What occurs in the phosphotransferase system?
The solute is chemically modified during passage through phosphorylation. The sugar phosphate backbone becomes negative and becomes trapped inside the cell where it can feed directly into catabolic pathways.
145
What do phosphotransferase system allow?
A fast growth rate as the first step of metabolism has already been completed.
146
Is the phosphotransferase system ATP driven?
No.
147
What are the four main components of the phosphotransferase system?
Enzyme 1, Enzyme 2, Enzyme 3, Hpr.
148
Which component of the phosphotransferase system is membrane bound?
Enzyme 2.
The rest are cytosolic.
149
What is the role of enzyme 1 in the phosphotransferase system?
Uses phosphoenol-pyruvate form glycolysis as a phosphate donor to self phosphorylate.
150
What is the role of Hpr in the phosphotransferase system?
Phosphorylated by Enz1.
151
What are the roles of Enzyme 2 and Enzyme 3 in the phosphotransferase system?
Transfers the phosphate to the sugar during transport.
152
What two components of the phosphotransferase system are cytoplasmic/soluble proteins which donate sugars to all sugar specific membrane bound transferases?
E1 and Hpr.
153
Why are the transporter domains different in different bacteria?
Evolution.
154
If the A + B transporter domains are both soluble what are they called?
Enzyme 3.
155
If the A + B transporter domains are membrane bout what are they called?
Enzyme 2.
156
Why must some proteins be transported and targeted across the membrane?
As many proteins have functions outside the cytoplasm.
157
What are the possible translocation mechanisms across the inner membrane?
The SEC and TAT system.
158
Why must the SEC and TAT mechanism ensure there isn't membrane leakage?
To prevent ion leakage.
159
Does SEC or TAT involve folder protein?
TAT.
160
Where is the SEC system found?
Euk and bacteria cells.
161
What is SecB?
A chaperone protein which binds to the nascent protein to prevent folding.
162
What forms the Sec pore which allows the threading of the protein?
SecYE.
163
What is SecA?
An ATpase which drives the folded protein.
164
What happens once the protein has been transported in the Sec system?
The N terminal sequence is cleaved by a periplasmic signal peptidase.
165
What does the transported protein in the Sec system contain?
The stop signal made of two K residues at the N terminal.
166
What end of the protein inserts into the membrane in the Sec system?
N.
167
What is found at the C terminus of a protein transported in the sec system?
AXA signal sequence.
168
What is an accessory protein in the Sec system?
SecG.
169
What is the prosthetic group of cytochromes?
Haem.
170
What is the prosthetic group of nitrate reductase?
Molybdenum cofactors.
171
What is the only periplasmic protein with an associated cofactor to be exported via sec?
Cytochrome C.
172
What sequence does cytochrome C use to insert into the membrane?
CXXCH.
173
Haem is exported from the periplasm separately to cytochrome C. How is it reattached to cytochrome C?
Covalently via two cysteines by specialised ligase enzyme.
174
Where is the TAT system found?
Bacteria, archaea and plant chloroplasts.
175
What does TAT stand for?
Twin Argine Translocase.
176
What do proteins transported via the TAT system contain?
A signal sequence with an invariant twin arginine (RR) motif.
177
What are the TAT proteins?
A, E, B.
178
What two TAT proteins are very similar so are probably functionally equivalent?
A and E.
179
What is the signal peptidase cleavage site found in proteins exported though TAT?
AXA.
180
What are the steps of the TAT mechanism?
1. The signal sequence is recognised and binds to the TatBC complex.
2. TatA then assembles onto TatBC and forms a large pore complex big enough to accommodate the folded protein.
3. Translocation occurs via the PMF.
4. Signal sequence is cleaved and the TatA and TatBC complex dissociates.
181
Does the TAT mechanism need ATP?
No it is driven by the PMF.
182
How many subunits of TatA are involved in the TatA mechanism?
Depends on the size of the protein. Protomers (subunits) are added as needed.
183
What cofactors does hydrogense have?
Fes, Ni-Fe.
184
What cofactors does formate dehydrogenase have?
Fes, MoCo.
185
Name an example of a cofactorless protein.
Cell wall amidases.
186
Transport of virulence protein in pathogens via the TAT method also requires what?
Secretion proteins.
187
What can be secreted through the TAT method from pathogens?
Some phospholipase and some proteases.
188
What type of extracellular enzymes can bacteria secrete?
DNases and Proteases which can act on lipases or toxins.
189
What periplasmic transport system uses the PMF and ATP for transport?
Sec.
190
What periplasmic transport system uses only the PMF for transport?
TAT.
191
What is exported in the type 1 system?
HylA (haemolysin VF).
192
How big is HylA?
107kda.
193
What are the three components of the Type 1 systems?
ABC transporter, large multi domain, outer membrane pori like protein.
194
What is the protein name of the ABC transporter in the Type 1 systems?
HlyB.
195
What is the protein name of the large multi domain in the type 1 systems?
HlyD.
196
What is the protein name of the outer membrane porin like protein in type 1 systems?
TolC.
197
What recognises the C terminal region of HlyA in type 1 systems?
HlyB.
198
Where is ATP hydrolysed in the Type 1 system?
HlyB.
199
What is translocated in type 1 systems?
The whole signal sequence- there is no cleavage of the signal sequence.
200
What do type 2 systems secrete?
Many extracellular enzymes and toxins such as the cholera toxin.
201
What membrane is the type 2 system found in?
Outermsmbrane.
202
What drives type 2 systems?
ATP.
203
What needs to happen before a type 2 system can be used?
The protein must be imported into the periplasm via Sec and TAT.
204
How many proteins make up a type 2 system?
12-15.
205
What is the type 2 system thought to be related to?
A bacterial pilus.
206
What are type 2 systems also called?
Sec dependant.
207
What are type 3 systems also called?
Contact dependant.
208
Why are type 3 systems important?
Many bacterial pathogens use them to directly inject pathogens into a eukaryotic cell.
209
How do type 3 systems inject pathogens into eukaryotic cells?
Injectisome.
210
How many proteins are type 3 systems made from?
17-25. The function of these are not well understood.
211
What does the structure of type 3 systems resemble?
Flagella basal body.
212
What happens when a type 3 system contacts a host cell?
The needle subunits assemble and protein translocation though the neck, bulb and needle begins.
213
What drives type 3 systems?
ATP hydrolysis.
214
What proteins can be delivered through a type 3 system?
Toxins or effector proteins.
215
What drives type 4 systems?
ATP.
216
What ca be transported in type 4 systems?
Protein or DNA.
217
What are type four systems involved in?
Bacterial conjunction and in some pathogens for protein secretion.
218
Where are half the genes encoding for type 4 systems found?
Pathogenicity islands.
219
What are type 4 system proteins related to?
Pilus proteins.
220
What are two examples of bacteria that use type 4 systems?
1. Agrobacterium tumefaciens.
| 2. Heliobacter pylori.
221
What does Agrobacterium tumerfaciens use type 4 systems for?
Transfer of plasmids between cells.
222
What does Heliobacter pylori use type 4 systems for?
Transfer of the car protein to the host cytoplasm allowing the cytoskeleton to be disrupted.
223
What are type 5 transporters also called?
Autotransporters.
224
How does transfer occur in a type 5 system?
C terminal of secreted protein forms a beta parallel porin like structure in the OM. The rest of the protein (passenger domain) is then transported through this.
225
What happens after transfer in a type 5 system?
The passenger domain is cleaved by a protease and o released into the medium.
226
How does the protein initially reach the periplasm in a type 5 system?
The SEC system.
227
What can type 5 systems be used for?
Some bacterial virulence factors such as IgA protease and other types of protease.
228
Where were type 6 transporters recently discovered?
In various gram negative pathogens.
229
What system does the type 6 transporter use and what is this related to?
A syringe like system which is related to the phage injection apparatus.
230
What are type 6 transporter systems known to do?
'Scratch' the OM of bacteria and deliver enzymes that dissolve in the peptidoglycan.
231
What bacterial strain uses type 6 systems?
Pseudomonas aeruginosa T655 which kills its completion by lysis.
232
What drives type 6 systems?
ATP.
233
In what system is it not known is it is driven by ATP?
5.
234
What systems move unfolded proteins?
1, 2, 5
235
What is the only system that can move DNA?
4.
