Lecture 7 Lipid-Protein Interactions & Membrane Dynamics

What is the fluid mosaic?

Pattern formed by individual lipids and protein units in a membrane, able to reorganize while maintaining its permeability barrier.

How is the lipid bilayer stable in water?

- Amphipathic lipids self-assemble in water.

- Hydrophilic head groups remain exposed to water.

- Hydrophobic tails cluster together away from water.

- This reduces exposure of hydrophobic surfaces to water.

- Bilayer can close into vesicles to eliminate exposed edges.

How does the endomembrane system exchange material and where?

Vesicle trafficking. ER, Golgi apparatus, endosomes, lysosomes, transport/secretory vesicles, plasma membrane, nuclear envelope.

How is the endomembrane system connected?

Budding, transport, and fusion of vesicles.How are the parts different?

How are the parts of the endomembrane system different?

Each compartment has distinct lipid and protein composition.

What is the purpose of the endomembrane system?

Organization allows cells to sort, modfiy, and deliver proteins and lipids to specific destinations.

What is special about lipid transfer proteins?

- LTPs contain a hydrophobic lipid-binding pocket that shields lipid tail from aqueous cytosol, allowing lipids to move between membranes without vesicle budding or fusion.

- LTPs act as membrane contact sites, where two organelles are close together.

- Some LTPs are bispecific, recognizing both specific lipid cargo and specific donor/target membrane.

What are examples of LTP and their function?

- CERT: Delivers ceramide from the ER to the Golgi for sphingomyelin synthesis.

- OSBP: Transfers cholesterol from ER to Golgi while PI4P moves back to ER.

- ORPS/ORP8: Helps enrich phosphatidylserine (PS) at the plasma membrane and regulate PM PI4P/PI(4,5)P2 levels.

- Nir2/PITPNM1: Transfers phosphatidylinositol (PI) to the plasma membrane and phosphatidic acid (PA) back to ER during phosphoinositide signaling.

What are the types of LTPs and how they function?

- Monomeric LTP: Single soluble transfer protein binds one lipid molecule from donor membrane to deliver to another membrane.

- Oligomeric LTP: Multiple protein units form larger complex between two nearby membrane, making lipid transport more efficient at membrane contact sites, where two organelles are close together but not fused.

- ATP-drive lipid movement: ATP hydrolysis can power lipid transfer or translocation when passive transfer would be unfavorable.

What are the types of lipid movement in the bilayer?

- Lateral diffusion.

- Uncatalyzed flip-flop.

- Catalyzed trans-bilayer movement.

What occurs in uncatalyzed lateral diffusion?

Lipids move sideways within same leaflet of bilayer, either in inner leaflet or outer leaflet. It will not cross through hydrophobic core. Movement is fast.

What occurs in uncatalyzed transbilayer diffusion (flip-flop)?

Lipids try to move from one leaflet to the other, where the polar head would have to pass through the hydrophobic interior of the membrane. This is energetically unfavorable. Movement is slow.

What are the proteins that help lipids move between leaflets in catalyzed transbilayer translocation?

- Flippase: Consumes 1 ATP per lipid molecule translocated. Moves specific phospholipids from outer leaflet to cytosolic leaflet. Helps maintain membrane assymmetry.

- Floppase: Uses ATP. Moves phospholipids from the cytosolic leaflet to the outer leaflet. Often belongs in ABC transporter family.

- Scramblase: Often activated by Ca2+. No ATP required. Moves membrane phospholipids non-selectively in both concentrations down concentration gradient. Important during processes like apoptosis, when phosphatidylserine becomes exposed to cell surface.

What are the different membrane lipid compositions?

ER: Enriched in glycerophospholipids; relatively low sphingolipid/cholesterol.

Golgi/Trans-Golgi Network: Sphingolipids and cholesterol increase.

Plasma membrane: Enriched in cholesterol and sphingolipids.

How do lipids recruit proteins to membranes?

- Integral membrane proteins are embedded in bilayer, like receptors, ion channels, transporters.

- Peripheral proteins bind membrane surface through weak interactions, like electrostatic interactions with charged lipids.

- Docking platforms have specific lipids recruit specific signaling proteins, like how PIP3 recruits Akt/PKB and PDK1 through PH domains.

- Lipid modifications can anchor proteins to membranes, like Ras prenylation, Src myristoylation, GPI-anchored proteins.

- Lock-and-key lipid recognition have protein domains bind specific lipid head groups, like how FYVE domain binds PI3P and PH domains bind PIP2 or PIP3.

How do lipids and proteins shape membranes?

Lipid shape affects membrane curvature: Cylinder-shaped lipids favor flat bilayers, cone-shaped lipids favor curvature, inverted cone-shaped lipids favor opposite curvature.

Mixed lipid shapes can create packing defects, which expose small hydrophobic regions for proteins. Amphipathic helices, like ALPS motifs and epsin ENTH domain, insert into packing defects.

BAR-domain proteins bind curved membranes and can stabilize/generate curvature.

What are the different ways integral membrane proteins associate with the bilayer?

Bitopic proteins cross the membrane once. Polytopic proteins cross the membrane multiple times. Monotopic proteins do not cross the whole bilayer, but insert partly into one leaflet.

What is the structure of a bitopic protein?

They span the membrane once using a single hydrophobic alpha-helix, while rest of protein remains exposed to aqueous environment on either side of membrane. Amino terminus is on outside, while carboxyl terminus is on inside.

What are the types of monotopic proteins and their degree of membrane insertion?

- CPT-II: 1% embedded.

- COX2: 7.6% embedded.

- PlsC: 16.2% embedded.

What does a monotopic protein do?

Interacts with only one leaflet of the membrane, not crossing the entire lipid bilayer.

What does a polytopic protein do?

Crosses the lipid bilayer multiple times with each region composed of about 20 hydrophobic amino acids that form an alpha-helix, which remain stable inside the hydrophobic core of the membrane.

What is an example of a polytopic membrane protein?

Bacteriorhodopsin crosses membrane seven times with each colored helix being one transmembrane alpha-helix. They are mostly hydrophobic on outside, allowing them to interact with fatty acid tails of bilayer. Loops connecting to helices are exposed to aqueous environment on either side of the membrane, while the amino terminus and carboxyl terminus are on opposite sides or specific sides depending on protein topology.

What are integrins?

Surface adhesion proteins that mediate cell’s interaction with extracellular matrix and with other cells, carrying signals in both direction across the plasma membrane. They are composed of heterodimeric proteins composed of two unlike subunits, alpha and beta.

What are cadherins?

Involved in surface adhesion. They undergo homophilic interactions with identical cadherins in an adjacent cell.

What are selectins?

They have extracellular domains that bind specific polysaccharides on the surface of an adjacent cell, mediating transient cell-cell adhesion, especially leukocyte rolling during inflammation. They usually require Ca2+.

How do peripheral proteins bounded and removed from the bilayer?

They bind through weaker interactions, like ionic/electrostatic interactions, hydrogen bonding, and interactions with other membrane proteins.

They are removed by treatments, like change in pH, chelating agents, urea, and carbonate.

What states can amphitropic proteins exist in?

Soluble in the cytosol or associated with the membrane. This is reversible (protein shuffling).

How can amphitropic proteins bind membranes?

- By recognizing specific membrane lipids, such as phosphoinositides like PIP2, PIP3, or PI3P.

- By reversible post-translational modifications, such as phosphorylation or palmitoylation.

- By covalent lipid anchors, such as myristoylation or prenylation. GPI anchors attach proteins to the outer leaflet and can be cleaved by phospholipases.

- By interacting with membrane-associated proteins or glycoproteins.

What are types of reversible post-translational modifications?

Phosphorylation regulates membrane association indirectly by changing protein shape, charge, or protein-protein interactions (MARCKS and many signaling proteins). Kinases add phosphate, and phosphatases remove phosphate. In MARCKS, unphosphorylated protein binds acidic lipids through positively charged residues. Phosphorylation often weakens that interaction by adding negative charges.

Palmitoylation attaches a palmitate, C16 fatty acid, to cysteine residue using palmitoyl acyltransferase, PAT (H-Ras, N-Ras). It is a reversible lipid attachment that helps proteins move on and off membranes.

What are types of covalent lipid anchors?

Myristoylation attaches myristate, C14 fatty acid, to N-terminus (Src-family kinases, ARF1). It anchors proteins to cytosolic leaflet.

Prenylation attaches a farnesyl or geranylgeranyl group to C-terminal cysteine (Ras proteins). It targets proteins to membranes.

GPI anchors attach proteins to the outer leaflet of the plasma membrane (alkaline phosphatase, CD55). These proteins face the extracellular space and cluster proteins in specific membrane regions.

How does photoconversion work?

It marks a small population of molecules at one membrane region. Before photoconversion, protein is seen in green. After local photoconversion, region becomes magenta. Over time, magenta signal spreads along membrane, showing lateral diffusion of membrane-associated molecules.

What are certain lipids and proteins that cluster into membrane domains?

Lipid rafts are dynamic, cholesterol- and sphingolipid-enriched membrane regions that are more ordered than the surrounding membrane and help concentrate signaling proteins.

Caveolae are flask-shaped plasma membrane invaginations, enriched in cholesterol, sphingolipids, and caveolin. They are involved in mechanosensing, endocytosis, and signaling.

Nanodomains are very small, short-lived membrane domains that can organize receptors, signaling proteins, and lipid-anchored proteins.

How do sphingolipids and cholesterol cluster together into membrane rafts?

The two pack well together because sphingolipids often have long, saturated fatty acid chains, which are straighter and pack tightly. Cholesterol fits between these straight lipid tails, creating a membrane region more ordered, thicker, and less fluid than surrounding phospholipid-rich membrane. They are separated into small local regions: More ordered raft-like domains and more fluid non-raft membrane.

What types of proteins can partition into domains on the membrane rafts?

GPI-anchored proteins on the outer leaflet, doubly acylated proteins like myristoylated + palmitoylated proteins, caveolin in caveolae, and some signaling proteins.

What are caveolae?

Small, flask-shaped invaginations of the plasma membrane considered specialized raft-like domains because they are enriched in cholesterol, sphingolipids, caveolin proteins, and cavin proteins.

What does caveolin do?

Membrane-associated/integral membrane protein that sits on cystolic side of plasma membrane that inserts into or associates with inner leaflet of the membrane, forms dimers/oligomers, binds cholesterol-rich membrane regions, helps bend membrane inward, and helps generate the curved shape of caveolae.

What is the function of caveolae in mechanical stress response?

Under low membrane tension, caveolae exist as membrane invaginations. When membrane tension increases, caveolae can flatten, providing extra membrane surface area and helping protect cell from mechanical stress.

How do nanodomains organize local signaling?

Lipids and proteins are mixed and mobile in the resting membrane, where some cluster into nanodomains. These areas will be enriched in cholesterol, sphingolipids, receptors, and lipid-anchored signaling proteins. They help concentrate signaling molecules in one local region.