Lecture 2: Membrane Proteins:

Membrane Proteins:

- Can interact with the lipid membrane in various ways

o Transmembrane

o One-sided

o Lipid-anchored

- Can have many different types of functions

o Signaling receptors (Transmembrane)

o Intracellular signaling (Lipid-anchored)

- Can move across lipid membrane

Transmembrane Proteins:

- Amphipathic

o Polar region outside of lipid membrane

o Non-polar region within membrane

- Glycosylated

- Single or Multi-pass:

o Single-pass

¡± Passes through the lipid membrane once

¡± Often a single a-helix

o Multi-pass:

¡± Passes through the lipid membrane multiple times

¡± Can be multiple a-helices or rolled-up b-sheets (b-barrel)

¡P More rigid

Membrane Proteins on One-side:

- Stuck to one side of the lipid-membrane

- Anchored by an amphipathic a-helix

Lipid-anchored Membrane Proteins:

- Synthesized in cytosol

- Faces the cytosol

- Protein can be connected to the lipid layer by:

o Fatty acid anchor

¡± Palmitate or myristate attached to N-terminal glycine

o Prenyl anchor

¡± Farnesyl or geranlgeranyl attached to C-terminal cysteine

Membrane Proteins attached through Non-Covalent forces:

- Attach to membranes by intermolecular forces with other membrane proteins

- Two types:

o Peripheral membrane proteins

¡± Can be extracted gently without destroying lipid membrane

o Integral membrane proteins

¡± Can only be extracted by destroying/solubilizing lipid membrane

Lateral Diffusion of Membrane Proteins:

- Can be sensed through Fluorescence Recovery After Photobleaching (FRAP)

o Fuse protein with GFP and bleach small area with laser

o Measure rate of GFP migration into the bleached area

o Allows observation of how fast the protein moves

o Only works if protein is free to move

Asymmetric Distribution of Membrane Proteins:

- Distribution of membrane proteins can be limited when cells form tight junctions

o i.e. Protein A will not be able to move to lateral and basal plasma membrane due to physical barrier

Source: http://www.cytochemistry.net/Cell-Biology/membr12.jpg

Cell Membranes:

- Permeable to gases, small (uncharged) polar molecules, most non-polar molecules

o These molecules can pass through simple diffusion

- Impermeable to large (uncharged) polar molecules, ions, and charged molecules

- Small organic polar molecules and inorganic ions can be transported through the membrane by transport proteins

o Example of facilitated diffusion

- Maintains cellular solute concentrations, which is important to cellular function

Membrane Transport Proteins:

- Multi-pass transmembrane proteins

- Transport polar or charged molecules (i.e. nucleotides, sugars, ions)

- Can be different for different types of cell membranes

- Each is specific to a type of molecule (i.e. glucose, K⁺)

- Have many different types of mechanisms

- Two types: Carrier Proteins and Channel Proteins

Carrier Proteins:

- Transporters, permeases

- Can undergo conformational changes when bound to specific substrate

- Transports substrate across membrane

o One molecule transported

Source: http://mws.mcallen.isd.tenet.edu/mchi/ipc/Biology/bioch03htm/bioimages03/FacititatedDiffusion_small.jpg

Channel Proteins:

- Pores across membrane

- Most are gated

o Regulated by chemical/electrical signal

o When opened, solutes pass through

- Have relatively weak interactions with solutes

- Migration through membrane is quick relative to carrier proteins

o Several molecules can pass through at once

Source: http://www.ifisiol.unam.mx/Brain/gifs/ionchnl2.jpg

Passive Transport:

- Follows concentration/charge gradient

o (Concentration + charge) = electrochemical gradient

- Does not require energy

- Spontaneous process

Active Transport:

- Moves against electrochemical gradient

- Requires energy (not always ATP)

- Non-spontaneous process

Note:

1) Charge gradient does not affect uncharged molecules

2) Both concentration and charge gradients affect charged molecules

Transportation by Carrier Proteins:

- Passive Transport (Uniporter):

o V_max is dependent on number of carriers are available

o K_m = ½ V_max

¡± Higher K_m = lower affinity of solute to carrier

¡± Influences the rate of transport

- Active Transport:

o Require energy

o 3 Types: Coupled Carriers, ATP-driven pumps, Light-driven pumps

¡± Coupled Carriers (Antiporters/Symporters):

¡P One molecule down gradient, another against gradient

¡P Example of secondary active transport

¡± ATP-driven pumps (or ATPases)

¡P Uses ATP hydrolysis (often to phosphorylate carriers)

¡P Example of primary active transport

¡P Three types: P-Type ATPases, V-Type ATPases, ABC Transporters

¡± Light-driven pumps (bacteria)

¡P Uses light energy to move molecule against gradient

¡P Example of primary active transport

- Uniporters, Symporters, and Antiporters:

o Uniporters:

¡± Passive transport

¡± One molecule down gradient

o Symporters:

¡± Active transport

¡± Coupled Carriers

¡± Both molecules move towards the same direction though one is down and the other is against electrochemical gradient

¡P Molecule going down gradient will provide energy for the carrier to bring the other molecule against gradient

o Antiporters:

¡± Same as symporters, but molecules go towards opposite directions

Examples of Symporter:

- Na⁺/glucose symporter

o Located in apical domain

o Used to transport glucose from intestinal lumen to blood

o Na⁺ goes down electrochemical gradient into the cell

o Glucose goes against electrochemical gradient into the cell

o Cooperative binding of Na⁺ and glucose

¡± Na⁺ is later pumped out by Na⁺-K⁺ ATP-driven pump

¡P Na⁺-K⁺ ATP-driven pump maintains low Na⁺ levels

¡± Glucose is transported out by GLUT uniporter

¡P Located in basolateral domain

Examples of Regulation of pH by Carrier Proteins:

- Antiporters:

o i.e. Na⁺-H⁺ antiporter:

¡± Na⁺ down gradient

¡± H⁺ against gradient

¡± Regulated by cytosolic pH

- ATP-driven pumps:

o i.e. ATP-driven H⁺ pumps:

¡± Used to maintain low pH in vacuole and lysosome

Na⁺/K⁺ pump:

- P-Type ATPase

- Phosphorylated by ATP hydrolysis

- Na⁺ and K⁺ move against electrochemical gradients

- Na⁺ gradient is used to transport nutrients into cells (Recall: Na⁺/glucose symporter), regulate pH (Recall: Na⁺-H⁺ antiporter), and cell volume

- Reversible è If Na⁺ and K⁺ concentrations are high, you can make ATP¡¦s

- Mechanism:

o Initially:

¡± Has 3 high-affinity Na⁺ sites (Na⁺ binds) and 2 low-affinity K⁺ sites (K⁺ binding dissociates)

o Step 1:

¡± 3 Na⁺ ions bind from cytosol

o Step 2:

¡± ATP hydrolysis occurs and binds to aspartate, forming a high-energy bond

o Step 3:

¡± Conformational change

¡P Na⁺ ions exit to extracellular space due to lowered affinity in Na⁺ binding sites

¡± K⁺ binding sites increase in affinity for K⁺

o Step 4:

¡± 2 K⁺ ions bind from extracellular space

o Step 5:

¡± Aspartate is dephosphorylated

o Step 6:

¡± Original conformation is re-established

¡± K⁺ ions exit to cytosol

o Cycle continues

Ca²⁺ ATPase:

- (P-Type ATPase)

- Pumps Ca²⁺ out of cytosol

- Maintains low Ca²⁺ concentration in cytosol

- Important for signaling (i.e. phytoalexin?)

- Found in sacroplasmic reticulum (SR) membrane of skeletal muscle

o SR stores calcium and Ca²⁺ pumps Ca²⁺ into its reservoir of Ca²⁺

Summery of Carrier Proteins:

1) Uniporters:

a. Carry out passive transport

2) Symporters and Antiporters:

a. Carry out active transport

b. Coupler carriers

3) ATP-driven pumps:

a. P-Type ATPases

i. i.e. Ca²⁺ ATPase, Na⁺/K⁺ pump

b. V-Type ATPases

c. ABC Transporters