Membranes and Membrane Proteins Flashcards
Biomembrane properties and membrane proteins. Insertion of Proteins into membranes
What are the basic components of membranes?
Sterols, Lipids (phospholipids, phosphoglycerides, sphingolpids), Proteins
What’s the structure of a lipid?
What determines their melting point?
Long hydrocarbon hydrophobic tail with polar carboxyl head (amphipathic– thus spontaneously forms lipid bilayers in aqueous solution.
Melting point increases with chain length but decreases with unsaturation.
Properties of biomembranes: Fluidity
- what affects this
- how can you measure fluidity?
- rapid lateral diffusion (btwn leaflets)
- slow transverse/flip-flop movement (through leaflets)
- composition-dependent: fatty acid length, type of lipid (# double bonds), steroids(eg, cholesterol decreases fluidity), proteins, temperature (but we’re at constant temp)
- measured using FAP (Fluidity After Photobleach): label the membrane protein with fluorescent reagent (fluorophore marker), bleach with laser a portion of cell, then measure fluorescence recovery (it’ll go back up, recovering from the bleach but only up to 50% of what it was, and there will be protein movement so you’ll see bleached spots in other areas than OG spot)
○ Tells us that yes, it’s a fluid system, but there’s also specific interactions that limit movement (ex, diffusion is 10X slower in plasma membranes with proteins than those without)— also some proteins are not mobile
Properties of biomembranes: Closed compartments
- Closed compartments: they limit not only the content of the pm but also they cover different the organelles in the cell.
Two leaflets in the lipid bilayer:
A. Cytosolic Face — internal face for plasma membrane, external face for vesicle membrane (think of how vesicles are made)
B. Exoplasmic Face – outer layer of pm
Properties of biomembranes: Semi-permeability
- small uncharged/hydrophobic molecules, gases can pass freely (water and urea are slightly permeable)
- large, charged/hydrophilic molecules, ions, large uncharged (glucose, fructose) are precluded (need another mechanism)
Properties of biomembranes: Asymmetry
- phospholipid composition differs btwn the two leaflets
- Glycans/carbs/sugars are only found in exoplasmic layer! ==> make up the Glycocalyx. (basically there’s a carbohydrate layer on the outside of cells)
- there’s a specific orientation for all proteins in the bilayer
Integral Membrane Proteins
- they span the pm; are asymmetric w/ 3 different domains:
Cytoplasmic Domain:
- hydrophilic
- charged AAs (Arg or Lys) are usually near cytosolic side to interact with polar head groups of the bilayer
Transmembrane Domain:
= hydrophobic 2ndary or 3iary structures that span the lipid bilayer
= generally an alpha helix or beta barrel
Exoplasmic Domain:
- most are glycosylated (have glycans on them)
Lipid-Anchored Membrane Proteins
= proteins anchored to membrane by lipid anchors (3 different types can be used:)
- GPI anchor: in exoplasmic side, needs sugar residues
- Fatty acyl anchor: in cytoplasmic side; attaches to N-terminal Glycine residue by acylation
- Prenyl anchor: in cytoplasmic side; attaches to Cysteine residue @ or near the C-terminus by renylation
- you’ll use Fatty acyl vs Prenyl based on how you want to insert the protein into pm – N or C first
- the actual polypeptide chain does not enter into pm
- has lateral mobility in membrane
Peripheral Membrane Proteins
- Not embedded in bilayer.. instead are attached to proteins that are in it via non-covalent interactions (ionic, H, protein-protein, van der Waals)
- cytoskeleton filaments (on inside, example Dystrophin), as well as ECM components can interact with bilayer by associating with these peripheral proteins (act as adaptors)
Insertion of Proteins into Membranes
5 Topological classes of integral proteins are synthesized on the RER
- classified by: orientation in the membrane (depending on if N or C terminal is in Cytosol or in Lumen of ER), and type of signal (topogenic) sequences they contain to direct them there
- also a difference: # of domains
- their final orientation is established during their biosynthesis on the ER membrane
Integral Proteins Type I, II, III, IV
Type I: NH3+ in exoplasmic space(lumen of ER), has a cleaved signal sequence attached to it which extends past NH3+
Type II: NH3+ in cytosol
Type III: NH3+ in exoplasmic space but like barely, most of the protein is in cytosol.
Type IV: NH3+ in exoplasmic space, but has tons of domains in pm! (7)
Tail-anchored: NH3+ in cytosol
Insertion of Tail-anchored Integral Proteins into ER membrane
- NH3+ in cytosol
- have NO extracellular domain!
- synthesized in cytosol
1) Get3-ATP binds the hydrophobic C-terminal tail of the protein
2) Get3-ATP-protein complex docks onto Get1/Get2 receptor on ER membrane
3) ATP is hydrolyzed and the tail becomes embedded (inserted) into the ER membrane within the Get1/Get2 receptor
4) Get3 binds ATP and dissociates from the ER, and leaves this tail-anchored protein in membrane or ER (COO- end first)
Synthesis of Type I Integral Proteins
- cleaved N-terminal signal sequence (cut by signal peptidase) determines initial interaction: targets the NH3+ end into the ER (aka, toward the lumen of the ER)
- the synthesis of the protein (on the ER membrane) continues until it hits the hydrophobic domain: Stop-transfer anchor sequence (STA). Once it hits this, the movement of the polypeptide stops, and the STA domain moves laterally to plasma membrane and stays there, becoming anchored in the membrane
- the alpha helix has strong interaction with translocon (what the protein was going through), and this eventually stops movement.
- the rest continues to be synthesized, and this part (C-terminus) is now the cytosolic part of the polypeptide
Topogenic sequences
= Cleaved N-terminal signal sequence
= STA (Type I)
= SA (Type II and III)
= Hydrophobic C-terminus (for tail-anchored)
Synthesis of Type II and Type III Integral Proteins
Signal-anchored internal sequence (SA) directs the insertion of the nascent polypeptide. The orientation of Type II vs Type III is determined by the positively-charged AAs(which are kept in cytosol!!!, no matter what orientation).
Synthesis of proteins occurs in cytosol as usual, and the translation stops once it reaches the SA sequence. Depending on where the ++AAs are, that’s which side will stay in cytosol.
