• What is a lipid bilayer and its permeability?
Form bilayers- essential components
of cellular membranes
■ Hydrocarbon chains (in some lipids)-
energy stores
■ Intra- and intercellular signaling
events
• What does it means for a lipid bilayer to be asymmetric?
Lipid bilayer asymmetry refers to the difference between the lipid composition and/or physical properties of the two lipid monolayers that make up a lipid bilayer
-That means that the two sides of membrane are structurally and functionally different
happens over time
•What is membrane fluidity?
Lipids move in the
bilayer
■ Depends on membrane
composition and
temperature
-Transverse diffusion (flip-flop) is very slow
-Lateral diffusion is rapid
- What factors effect fluidity of membrane?
- Temperature-Real membranes have liquid-ordered domains (Lo) or “rafts” due, in part, to cholesterol, a membrane “plasticizer” and sphingolipids
- above transition temperature
- Ld: liquid-disordered stat
- Below transition temperature
- So: solid-ordered state
- above transition temperature
- Fatty acid chain length alters fluidity- shorter chain, closer to temp on graph
Experimentally how you can test membrane fluidity?
■ FRAP – Fluorescence Recovery After Photobleaching
- watch video
- Be able to read and draw FRAP data.
- What is the fluid mosaic model?
Biologists use the fluid mosaic model to describe a membrane’s structure—diverse protein molecules suspended in a fluid phospholipid bilayer (phospholipids, cholesterols, and proteins)
- Integral membrane proteins
- How can they be isolated from the membrane?
The isolation of integral membrane proteins requires more drastic conditions, and generally, detergents (also called surfactants) or organic solvents have to be used to extract the protein from the bilayer.
Contain transmembrane domains
(Integral membrane proteins) What amino acids would you expect to find spanning the membrane?
Nonpolar amino acids like Leucine
How do you read and interpret a hydrophobicity plot.
Watch video-
- Peripheral membrane proteins
- Describe how are they associated with the membrane and how can they be isolated from the membrane?
■ Bind to membrane surface – Electrostatic interactions – Hydrogen bonding
Adhere only temporarily
peripheral proteins are removed by shifting the ionic strength or pH of the aqueous solution, thereby dissociating the ionic interactions of the peripheral protein with either phospholipid polar head groups or other membrane proteins
- Lipid-linked proteins
- What is Prenylation?
■ CXXY motif
– X = aliphatic amino acid
– Y = Ala, Met, Ser à farnesylated
– Y = Leu à geranylgeranylated
Prenylated proteins have covalently attached lipids that are built from isoprene units
- Lipid-linked proteins
- What is the recognition sequences for addition of farnesyl and geranylgeranyl residue?
I cant find how it actually recongizes???
In general, the consensus prenylation sequence contains the CAAX motif (referred to as the CAAX box; C is cysteine, A is usually an aliphatic amino acid, and X can be a variety of amino acids). The X residue of this motif largely determines the choice of the isoprenoid
- Lipid-linked proteins
- Acylation
Be able to identify and know how they are linked
■ Myristolation – Myristic acid attached to Nt amino group of Gly
■ Palmitoylation – Thioester linkage to Cys
- GPI anchored proteins
- Be able to identify/describe the GPI anchor
- Where is the lipid modification added to a protein?
GPI anchor – Glycosylphosphatidylinositol- linked protein – amide linked to Ct
- Secretory system
- What are free and bound ribosomes and where proteins that are translated on each are distributed in the cell?
Free ribosomes – Soluble and mitochondrial-bound proteins
■ Membrane bound ribosomes – Secreted, transmembrane, lysosomal, and ER proteins
- Secretory system
- What is the process by which Type I transmembrane proteins are manufactured in eukaryotic cells?
Translation begins on a free ribosome
■ Signal peptide leads to halting of translation and localization to the rough endoplasmic reticulum
■ Ribosome binds translocon and translation continues
■ N-terminal signal peptide not part of final protein
■ Signal-anchor sequence transfers into rough ER membrane during translation
■ Post-translational modifications occur in the ER and secretory pathway
■ No signal-anchor: soluble protein
- Secretory system
- What is the functions of the translocon?
■ Allows passages of peptide sequences into the ER
■ Facilitates insertion of proteins into the membrane – Signal-anchor sequences
- Secretory system
- Describe the movement of COPI, COPII, and clatherin coated vesicles.
- What is the purpose of the protein coat?
- Describe the movement of COPI, COPII, and clatherin coated vesicles.
- Clathrin-coated vesicles transport TM, GPI-linked, and secreted proteins to the plasma membrane
- COPI-coated vesicles bud from the Golgi compartments
- COPII-coated vesicles bud from the ER
■ Vesicle coating
– Occurs after cargo binding
– Causes the membrane to evaginate
Vesicles cannot fuse to a membrane with coat bound
■ Vesicles also contain: – Targeting proteins – Fusion proteins
- Secretory system
- Describe v-SNAREs and t-SNAREs and how they facilitate membrane fusion.
Also, be able to describe their disassembly
R-SNARE (v-SNARE) and QSNARE (t- SNARE) zip together and cause membrane fusion
■ Vesicle and target membrane become continuous
Interior of the vesicle and outside of the cell are topologically equivalent
■ Fusion with membrane preserves the orientation of integral proteins
■ Upon fusing, cargo inside vesicles is released to the extracellular matrix
