Biological Membranes Flashcards
What are the three types of secondary transporters?
Uniporters, Symporters and Antiporters
What are Uniporters?
Like channels, a single substrate is transported each cycle
What are Symporters?
Both substrates go in the same direction. E.g leuT, GltPh, VcINDY
What are antiporters?
Substrates move in opposite directions.
What are antiporters and symporters known as?
Co-transporters
Describe Symporter alternating access mechanism?
Substrate binding site is alternated from one side of the membrane to the other.
1. Molecules bind in open facing state, substrate then binds
2. Once loaded, it transitions through the membrane to release substrates into the cytoplasm via its inward facing state.
3. Once empty, it moves back through the membrane, returning to outward-facing state.
What does alternating access maintain?
Tight coupling as it can only move through different conformational states when it is fully loaded or empty
What an example of the antiporter transport mechanism?
Ping-pong mechanism.
1. Substrate from cytoplasm binds to inward facing binding site.
2. Induces conformational change, flipping the transporter to its outer-facing state.
3. Substrate is released extracellularly.
4. This allows other substrates to bind. Once bound, this induces the inward facing state.
5. Transporter releases contents into cytoplasm. Allows substrate binding once again
Why are antiporters needed?
- Maintenance of Ionic Asymmetry
- Organelle Acidification
- pH Homeostasis
- Extrusion of toxic compounds
What are passive mechanisms of Adaptations to bacterial acid stress?
- Natural buffering capabilites of amino acids.
- Proteins
- Polyamines
What are the active mechanisms of adaptations to bacterial acid stress?
Physiological, Metabolic and Proton-consuming
What do physiological adaptations consist of?
- Modifications of the lipid bilayer.
How does modification of the lipid bilayer resist acid stress?
Enzyme produced that modifies unsaturated fatty acids into cyclopropane. Effects stability of the membrane.
Thickening of the membrane also does this, reducing ability of protons entering.
What is produced that assists modification of lipid bilayer against acid stress?
PolyP and Cadaverine - inhibitors of the outer membrane porins.
They reduce the amount of protons entering the cell.
What do proteins in the periplasm do to withstand acid stress?
Conformationally change. Chaperones can keep the proteins attached to them until the pH returns to 7, which then triggers them to refold.
How do metabolic conditions resist acid stress?
Protons are pumped out of the periplasm via a set of enzymes.
How does active H+ consuming resist acid stress?
Multiple codons in E.coli that encode enzymes and transporters, modify substrates to bind a proton to the substrate.
Pumps out proton as well as the substrate.
Which antiporters are involved in bacterial acid stress?
AdiC, GadC, CadB and PotE
What sort of mechanism is AdiC proposed to deploy?
Ping-Pong, binding of arginine and agmatine.
Steps involved in AdiC function?
Binding of arginine in outward facing state = isomerisation of the protein inwards. Leads to inward facing state conformation and release of substrate.
Reverse happens with agmatine
What happens when the outside is acidified with AdiC function?
Activity is increased
What prevents rebinding of arginine in AdiC?
The Outward facing state having a 5 fold reduced affinity for Agm. Changes in conformation reduce affinity.
Which transporters have been shown to have involvement in drug resistance?
MATES - multidrug and toxin extrusion transporters
Which was the first MATE to be discovered?
NorM
What makes NorM able to transport lipid soluble drugs?
The transport unit has a lipid bilayer accessible vestibule in it.
What is the mechanism of NorM hypothesized to be?
Allosteric Antiport mechanism. They have cations bound within them.
What is the universal lipid carrier?
C55P, attached to for flipping across the membrane
What do antiporters have involvement in to do withh bacterial structure?
Peptidoglycan synthesis
What is the process of peptidoglycan synthesis?
C55P flips precursors of peptidoglycan which makes NAM, leading to lipid 2 formation.
MurJ then flips Lipid 2 from inner to outer leaflet, where it is used by enzymes and added to peptidoglycyan.
C55P is then flipped and recycled to be used again.
What does MurJ have the same fold as?
MATES
What is different about MurJ?
Has hydrophobic groove and 2 other binding sites.
Chloride Ion in MurTA, not protons.
Sodium in MurEc.
Sodium ion site in binding sites of these is similar to PfMATE
What is the mechanism of MurJ transport?
- Tube in membrane, lipid B enters into the core this way. Tail fits into groove.
- Sodium binding induces conformational changes, and allows the head group of Lipid II to exit.
- Protein enters the outward facing transitional state.
- Chlorine rebinds and returns to the inward facing state.
What is MurJ?
Lipid 2 Flippase
MurJTa is ___ independent but ___ dependent.
Na+, Cl-
What disrupts lipids in a bilayer?
Scrambalase
Experiment showing lipid transport mechanisms?
- Tag lipids with fluorescence marker and add a reducing agent.
- In scrambling lipid, the action of protein flipping moves lipids from one side to the other = contact with fluorescence on both sides.
What does addition of Dithionite and Triton X 100 mean in lipid transport experiments?
Quenching of fluorescence, all lipids present will be exposed.
What is Dithionite?
Reducing agent used in lipid experiments
What does Dithionite cause in lipid experiments?
Reduction of lipids, quenching of fluoresence = reduction.
What are the 3 types of secondary transporter mechanisms?
- Rocker Switch - Moving Barrier
- Rocking Bundle - Moving Barrier
- Elevator - Fixed barrier
How do the mechanisms of secondary transport vary?
In position of the barrier, position of the binding site and differences in the type of barrier.
What is the aim of secondary transporter?
Bind to a substrate in one conformation and switch to a different composition to expose the binding site from one side of the membrane to the other.
What are examples of transporters in the synaptic cleft?
EAATs, GLUTs and NSS
What is the largest transporter family?
Major Facilitative Superfamily (MFS)