Membranes Flashcards
Membrane Components
Phospholipids
Glycolipids
Sterols
Factors affecting membrane fluidity
Length of hydrocarbon chain
Saturation of hydrocarbon
Sterols
Movement of synthesized lipids
Lateral, Spin, flip-flop
Evidence for flip-flopping
Label all phospholipid heads with spin-label
Add ascorbic acid to reduce spin-label on outer phospholipids, measure spin label
Incubate at 30oC, add ascorbic acid, and measure again - will be lower due to flip-flopping
Determining if protein is trans-membrane
Label liposome containing membrane protein with radioactive iodide by peroxidase-dependent iodination
Disrupt another liposome containing the protein, and label with peroxidase-dependent iodination
Run both on SDS-PAGE, and autoradiograph. Additional labelling in disrupted membrane means protein is transmembrane
Evidence for lateral movement of protein
Cell fusion of mouse and human
Use mAb conjugated with fluorophore to observe mixing of human and mouse proteins across membrane
FRAP
Treat cells with conA conjugated with GFP. Laser to bleach small area. Time return of fluorescence in area due to diffusion of glycoproteins
Membrane Protein Trafficking
Signal sequence recognized by SRP
Translation halted
SRP docks on SRP receptor; brings ribosome to ER
Translation resumed; polypeptide tunnels through into ER lumen
Signal sequence cleaved by signal peptidase
Integral protein has stop-transfer sequences allowing fixing and orienting in bilayer
Non-integral protein released into ER lumen, allowing folding by ATP-dependent foldases and chaperones
Secretory/Membrane pathway
Protein leaves ER in transport vesicles that bud from rib-free region of ER
Delivered to cis face of golgi
Pass through Golgi apparatus by cycles of vesicle budding and fusion
Glycosylation and Covalent modification
Sugar residues added to membrane/secretory proteins
Sugar added as oligosaccharide catalyzed by glycosyl transferase during passage into ER
Additional sugars added to form side branches in Golgi
