Membrane proteins Flashcards
What are the roles and components of the Secretory pathway?
Protein/Secretory pathway components synthesis in ER → transformed in the Golgi → secreted in plasma membrane, extracellular or other organelle of secretory pathway through vesicles
- INTERNALIZATION (enter the cell) through endosomes → fused to lysosomes
- Degradation in Lysosomes
*Never in direct contact with cytosol, spearated by membrane
Are all organelles connected/in contact with the secretory pathway?
No!
- Not mitochondrias
*The nucleus and cytosol are not in contact with the secretory pathway,
Secretory pathway includes: ER, Golgi, Exterior of the membrane
What is the Lumen?
Lumen = interior of the secretory pathway organelles → ER, Golgi,
- Continuity in components of all secretory pathway organelles
*Components of the lumen are never in contact with the ones of the nucleus/cytosol
Where are the membrane proteins synthesized?
All protein synthesis is done in the cytosol
How What is the lumen environment similar to? and not similar to?
Similar to extracellular space → blood plasma → salts, pH, proteins, cofactors
Different from cytosol
How is transport done from one component to another of the secretory pathway?
Through vesicle transport:
- plasma membrane of organelle forms a closed vesicle → Budding
- Travels in cytosol staying closed
- Releases its content to target organelle of the secretory pathway → fusion
What are the main differences between contents or the cytosol and of the lumen/extracellular space?
Which one can make disulfide bonds?
Cytosol = reducing environment, No disulfide bonds
Lumen = oxidizing environment (losing electrons), Disulfide bonds can be made
What are important functions of the biological membranes?
- Provide enclosure to the cell + to organelles within the cell
- Allow regulated transport between compartements
- Provide sites for biochemical reactions within the cell
- Suport contacts with enviroment outside the cells
What reactions are allowed in the cell by the fact that biological membranes provide possibility to have different environments?
Photosynthesis, oxidative phosphorylation (cytosol)
VS
Metabolism of biological molecules: lipids glycans, etc. (lumen)
What form of contacts with the outside environment of the cell are supported by the presence of biological membranes?
- Cell motion
- Recognition of other cells
- Cell fusion
- Transmission of signals from exterior to interior of the cell
What are the properties of biological membranes? (4)
- Form HYDROPHOBIC barriers between aqueous compartments within the cell (cytosol and organellar lumens)
- Flexible and can be formed into different shapes
- Selectively permeable
- Can store energy as concentration gradients: voltage (nerve cells), pH, K+, Na+, Ca++
What does it mean for the plasma membrane to be selectively permeable?
- Small hydrophobic molecules can transpass the barrier
- Hydrophilic and big hydrophobic molecules need transport proteins to cross
What are the structural components of membranes?
What is its model?
FLUID MOSAIC MODEL
2 components: Lipids and Proteins
- Lipids are organized into a bilayer → polar exterior and hydrophobic interior
- Hydrophobicity = barrier to water-soluble molecules
- Membrane proteins can rotate and diffuse laterally, but can NEVER flip
What are the 3 major lipids in the plasma membrane?
And Characteristics?
- Phospholipids → in all membranes
- Glycolipids → only in plasma membranes (no in ER or Golgi)
- Cholesterol → all membranes, preferentially in plasma membrane
All have a polar head and hydrophobic tails
Lipid composition determines physical proterties of membrane
→ mobility (diffusion, rotation, can flip from 1 layer to the other)
→ curvature, thickness
What is the general structure of phospholipids?
Polar head groups:
- Choline or other charged group
- Phosphate (1 negative charge)
- Glycerol
– Ester Linkage –
Hydrophobic/fatty acid tails:
- different lengths
- Saturated or unsaturated (1 or more double bonds)
- Found in many different combinations with head groups
Which is the most abundant lipid in membranes?
phospholipids
What are the different phospholipids charged head groups?
- Phosphatidyl-choline (PC) → (1 positive charge to neutralize phosphate)
- Phosphatidyl-ethanolamine (PE) → ( 1positive charge to neutralize phosphate)
- Phosphatidyl-serine (PS) → (1 positive charge + 1 negative charge so net negative charge)
- Sphingomyelin (SM) → same group as choline but has amide linkage tails like a glycerolipid, Ceramide basis
- Phosphatidyl-inositol (PI)
Head group size and charge affect lipid mobility
What is phosphatidyl-inositol?
A phospholipid head group
Not abundant but can be phosphorylated and act as signaling molecule
- Sugar attached to the phosphate, this sugar has -OH groups that can be phosphorylated
What are the characteristics of fatty acid tails?
- Hydrocarbon chain of 14 - 24 C
- Saturated or not (varying # of double bonds)
- Saturated → straighter, more flexible
- Double bonds (cis) → bends in the tail, less flexibility, shorter length
- Types of tails in membrane determine its thickness and fluidity (more double bonds = more fluidity)
*Synthesized in cytoplasm
What are glycolipids?
They are lipids found on the outside surface of the plasma membrane only
Structure:
Basis = ceramide (includes 1 fatty chain, 1 fatty acid tail + amide linkage)
Head group = glucose, GalNac, Gal, NANA sugars or combinations
head groups → important for contacts with environment and other cells
What is the role of cholesterol in membranes?
*Structually different from other lipids
Steroid ring structure → very rigid:
- lateral mobility, rotation → much lower
- Less mobility of surrounding phospholipids
- Makes fatty acid tails more rigid
What is the structure of cholesterol in membranes?
- Polar head group (-OH)
- Rigid steroid ring structure (stiffened region)
- 1 nonpolar hydrophobic tail (more fluid region)
How is the membrane asymmetric?
- Lipid composition at each side is different
- Important for PLASMA MEMBRANE function
- Exterior has glycolipids
- Interior → stronger negative charge (high PS levels)
- Not absolute asymmetry, but actively maintained by phosphorylation of phosphatidyls (usually in interior)
What are the main components of the outer leaflet and inner leaflet of the plasma membrane?
Outer leaflet (facing extracellular space):
- Phosphatidyl-choline (PC)
- Sphingomyelin (SM)
- Glycoplipids
Inner leaflet (facing cytosol):
- Phosphatidyl-ethanolamine (PE)
- Phosphatidyl-choline (PC)
- low amout of PI for signaling
Which lipids is the plasma membrane mostly rich in?
- Cholesterol
- SM
- PC
- PE
-GL → *Very little but only found in PM
Which lipids is the ER membrane mostly abundant with?
- PC
- PE
Which lipids is the mitochondria membrane mostly abundant with?
- PC
- PE
*Dosen’t have SM nor GL
What are microdomains?
Regions of a membrane that are organized laterally (sideways), in patches
What are lipid rafts?
Special micro domains in Plasma membrane and trans-Golgi:
- Enriched in Cholesterol → Thicker than surrounding membrane
- Lipids with longer tails cluster in rafts
- Different proteins content and biological functions
Why does cholesterol binding thickens the membrane?
It straightens the lipid tails
What is the polarity/hydrophobicity of cholesterol like?
Very hydrophobic because of the rings
*Has weak hydroxyl polar head
Name the lipids in increasing order of size.
And compare to protein and AA sizes
Cholesterol < Phosphorylation lipids < Glycolipids
AA < phospholipids < proteins
What are the types of membrane proteins?
Integral membrane proteins:
- Transmembrane Helices → Type 1 and 2
- Transmembrane barrels
Amphipatic alpha helix
Lipid anchorage proteins :
- Acylation and Prenylation
- Glycosyl-phosphatidyl-inositol anchor
Lipid anchorage via GIP
Peripheral membrane proteins
Explain phospholipid synthesis.
Synthesized on the cytosolic side of the ER membrane (not ER lumen side)
- Fatty acids (acyls) binding protein brings fatty acid-OH to the membrane
- Fatty acids are attched to CoA in chemically reactive states
- Glycerol-phosphate, head group added in sequence by enzymes
Where are phospholipids and cholesterol synthesized?
In the cytosolic side of the ER membrane
How does synthesis and insertion of lipids in the ER membrane occur?
- phospholipids + cholesterol synthesized in cytosolic side of ER membrane (Not ER lumen side)
- phospholipids add to cytosolic half of the bilayer → makes membrane unbalanced
- Scramblase catalyzes flipping of phospholipids → symmetric growth of both halves of bilayer
*lipids are transported by secretory pathway by vesicles
What is Scramblase?
Enzyme responsible for flipping of the phospholipids from cytosolic side of ER membrane to the ER lumen side
ATP-independent → functions bc of “concentration gradient” between both layers
How is asymmetry maintained in the PLASMA membrane?
*After delivery of new membrane by exocytosis/vesicles, lipids might not be in the right layer
Flippase proteins maintain membrane asymmetry:
- ATP-dependent
- directional
- specific phospholipids to cytoplasmic monolayer
PM NOT as random as ER
How does the transport of lipids occur?
Maintain lateral organization + membrane asymmetry
Lipids are transported by:
- Vesicles between organelles of the secretory pathway
- Carrier proteins through the cytosol
- through direct physical contact sites between organelles → ER and mitochondria
Which 3 main aspects of proteins are determined by their AA sequence?
- 3D structure → function
- Post-translational modifications
- Where the protein is located in the cell (in the membrane?, in which compartment? in the cytosol?) → proteins in the cytosol ≠ lumen ≠ membrane
What PTM do membrane proteins undergo?
Addition of lipid groups
- Acylation / prenylation
Soluble protein are NOT associated with membrane proteins
Are soluble proteins associated with membranes?
No, because they are polar and membranes are hydrophobic inside
What are 3 different integral membrane proteins?
What is common to all of them?
- 1 or more transmembrane alpha-helix
- Transmembrane beta-barrel → make a cylinder
- Amphipatic alpha-helix in one face of the membrane (doesn’t touch both side of the membrane) → hydrophobic on one side (membrane side) and polar on other (cytosol side), less abundant
*All tightly anchored by hydrophobic interactions with interior of the lipid bilayer
What are lipid-anchored membrane proteins?
Proteins that are covalently bound to one of more lipids or fatty acid groups on the membrane
- The strength of the anchor depends on number and type of the lipids its attached to
What are lipid-anchored and peripheral membrane proteins?
Lipid-Anchored:
- Covalently interacts with membrane
Peripheripheral membrane proteins:
- Attached by non-covalent interactions
- Have strong protein:protein interactions with integral membrane proteins → localizes them in the cell
- Or can have weak interactions with lipid head groups → localization role too
What are the main differences in functions between transmembrane proteins and lipid-anchored proteins?
Transmembrane proteins:
- Function in both cellular compartements (both ends)
- Cell surface receptors, transporters
Lipid-Anchored proteins:
- Function in one side of the membrane (either side though)
- Intracellular signalling
What are the characteristics of a transmembrane helix?
- Most common form of attachement
- Side chains point outwards and are hydrophobic to interact with lipids → G, A, C, L, F (important)
- Can have few polar groups on the ends to interact with polar head of lipids
- 18-24 AA long → longer than normal intradomain helix, longer for thicker membranes, angled for thinned membranes
- Protein with single TM can rotate easily
What amino acids form the side chains of transmembrane helices?
Glycine, Alanine, Cysteine, Leucine, Phenylalanine
What does the structure of a group of multiple TM helices looks like?
- Can for a channel/port for ions for example
- Can have hydrophilic side chains that make the port a hydrophilic environment
What is the structure and characteristics of transmembrane beta-barrels?
- beta-strands wrapped into a cylinder → TM barrel
- # TM strands can vary
- Side chains point out into lipids bilayer and inside
- H-bonding holds strands together
Why are membrane proteins never flipped?
Because the asymmetry in protein membrane configuration → cytosolic side doesn’t do same PTM as lumenal side (reducing vs oxidizing envrionments)
Modification in the lumenal/extracellular side:
- Disulfide bonds between cysteines
- addition of oligosaccharides (glycosylation)
→ Stabilize protein structure
Cytosolic modifications:
- Phosphorylation, Ubiquitination, Acetylation, Methylation
What are the differences between channels and pumps/transporters?
Chanels and pumps = Usually beta barrel or multi-pass (alpha helices) proteins
Channels:
- Mvt along gradeint
- No energy requirements
- Regulation = opening and closing
Pumps:
- Mvt against gradient
- ATP-dependent
- regulated by turning ATPase on/off
What are the characteristics of channel proteins?
Allow flow of ions (or other molecules) across membranes
TM helices can form water-filled channel → opening controlled by cytosolic domains/subunits
Selective Filter for specific ions :
- Carbonyls (=O) from peptide backbone line the pore (replace O from H2O they bind in the cytosol)
- Ions normally bind water → if right size for the channel → can exchange H2O for =O → partial dipoles
- If wrong size, can’t completely bind carbonyls → rejected
ex: voltage-gated K+ channel → neuron signaling, heart rhythm → controled by voltage sensors
What are ATP-Binding Cassette (ABC) transporters?
Give an example.
Large family of membrane transporter proteins
transport small molecules → cholesterol, toxins, phospholipid flippases
(against concentration gradient)
Multidrug resistance transporter:
- ABC transporter with 2 symmetrical ATPase domains
- pumps toxins out of cancer cells → resistance to chemotherapy
- NBD1/2 (nuclear binding domains) in cytosolic side (intracellular) + TMD1/2 inside the membrane
3 states:
1. No nucleotide → inward open, high affinity for substrate to come and bind
2. ATP bound → closed (with substrate)
3. ATP hydrolysis → outward open, low affinity for substrate (release outside of cell)
What PTM can undergo cytosolic proteins in order to become lipid-anchored?
What is required for strong membraen anchor?
Can be convalently linked to acyl (fatty acid) → acylation or phenyl chains → prenylation
- Single lipids chains provide transient interaction with membrane
- 2+ lipids chains need for strong membrane anchor
- Specific enzymes attach to N-terminus or Cys side(C-term) chain
What is required for Acylation of cytosolic proteins → lipid anchored)? What is the name of the bond?
N-terminus methionine has to be cleaved → Need Glycine or Cysteine as 2nd AA
Glycine or Cys → amide linkage
Cysteine anywhere → thioester linkage
G and C not involved in N-end rule, so works out
Need multiple acylations for strong membrane anchor
What is required for Prenylation of cytosolic proteins (→ lipid anchored)? What is the name of the bond?
Need a prenyl group at the end of protein:
motif = Cys-a-a-x-COO-
a = alkyl side chain, x = any
aax sequence cleaved off, Cys is prenylated
Thioether linkage with cystein (has S) and prenyl group
Which lipid modifications in the cytosol (acylation and prenylation) are permanent vs reversible?
Cys S-acetylation → reversible (thioester) → signaling (often done by kinases)
All prenylations are permanents
Acylations → Amide to N-Gly and Amide to N-Cys → Permanent
What PTM can Cysteine AA undergo? (Chemically reactive Sulhydryl side chain)
- Disulfide bond formation
- Ubiquitin E1 activating, E2 conjugating enzyme (thioester)
- Lipids modification (Thioester, Thioether) → Acylation
- Prenylation motif: Caax-COO-
What are GPI-anchored proteins?
Starts as transmembrane protein → Has special TM helix removed → covalently linked to GPI anchor → More rotational freedom than proteins with TM helices to move farther in ER lumen, staying attached to membrane
*GPI = glycosyl-phosphatidyl-inositol (anchored in the PM)
- Strong membrane attachement
- Only on lumenal/extracellular side
- Function at exterior of the PM (in the lumen)
What happens to the structure of an integral membrane protein in the absence of a membrane?
What happens to lipid-anchored and peripherial proteins?
Integral membrane proteins: different 3D structure because of its hydrophobic AA
Lipid-anchored and peripherial proteins: no change
Where is sorting information for each protein carried? Why is it important?
Sorting information is important because all proteins are encoded by nuclear genes, translated in cytosol
- Sorted during/after translation to their correct compartment
Sorting information is carried inside the proteins themselves
Where do sercretory pathway proteins go after/during translation?
- ER for ALL of them
- Transport to further compartements → Golgi, PM, endosomes, lysosomes
What are some characterstics/sections of ER?
Outer and inner nuclear membrane are continuous with ER
Rough ER = many attached ribosomes, secretory protein synthesis
Smooth ER = no ribosomes, sites of lipid synthesis
What are the properties of targeting signals on proteins?
- Sequence within a protein that specify its organelle localization → signal peptide
- Can also be a PTM
- Are often idependent from the structure or biochemical function of protein
- May be removed by proteolysis after targeting is complete, or form part of the native structure
- ## Recognized by their pattern, not usually exact sequence
What are the targeting steps for targeting proteins to their specific organelle localization?
- Recognize a signal on a protein:
- Ribosome begins translating polypeptide with a signal
- Signal Recognition Particle (SRP) = soluble protein that binds signal and ribosome during translation (at the exit tunnel) → Slows down translation by binding to ribosome
*SRP-GTP binds (not SRP-GDP) - Connect protein to the membrane:
- SRP Receptor (SRP-R) = membrane protein → binds ribosome-SRP complex (rlly only binds the SRP)
- SRP-R links ribosome to translocon pore in ER - Translocate protein into or across the membrane (in translocon):
- Energy of translation on ribosome drives polypeptide through the translocon
*For secretory signal peptides → CO-TRANSLATIONALLY
How were targeting signals discovered?
Observation: newly translated secretory protein larger than its final form
Hypothesis: extra sequence is a targeting signal peptide whose main function is to direct insertion into ER
- Signal peptide must start mechanism to connect ribosome to transcation pore
- Signal peptide is cleaved off after trageting is finished
What is the Ribosome Exit Tunnel?
Path, in large (60S) subunit by which nascent polypeptide exit the ribosome
- Neutral, polar, too small for tertiary folding
- Surface around exit site provides binding sites for ER targeting mechanisms
- 30-40 AA of nascent polypeptide between peptide-transferase site and the exit
What is the signal peptide pattern for proteins of the secretory pathway?
8-16 Hydrophobic AA + short polar regions on each side
- In most cases, at the N-terminus (beginning)
- Often cleaved off after translocation
What are signal anchors?
Signal peptides that also become TM helices:
- Not cleaved off
- Can be in different places in the protein
- longer hydrophobic region → 18-24 residues
Also bound by SRP-GTP for type 2 proteins to bring to translocon
Where do secretory pathway proteins start their translation?
IN THE CYTOSOL
Almost all proteins are coded for by nuclear genes and their mRNA is translated (start) in the cytosol with ribosomes
What is the structure/functions of SRP?
Signal Recognition Protein
- Ribonucleoprotein → 6 protein subunits + 1 RNA
- Signal sequence recognition subunit with GTPase activity
- Translation regulatory domain (slow translation down when binds to the ribosome)
- RNA strand = flexible linker
How does SRP interact with ribosomes?
- SRP samples ALL nascent polypeptides that emerge from ribosomes
- If signal peptide is recognized, SRP attached tightly to both the signal and the ribosome → pauses translation at ribosome and binds GTP
How is the ribosome transfered from the SRP-R the the translocon?
- Ribosome-SRP complex binds to SRP-R on ER membrane (cytosolic side)
- Ribosome moves to the translocon and becomes tighlty bound
- SRP and SRP-R dissociate from ribosome → translation resumes, polypeptide translocated into lumen, lumenal polypeptide never in contact with cytosol
How is GTPase activity involved in SRP and SRP-R’s roles in the targeting signal of secretory pathway proteins?
- SRP attached to ribosomes = GTP-bound state
- SRP-R (also GTPase) recognizes SRP-ribosomes = GTP-bound state
- GTP hydrolysis by both SRP and SRP-R dissociate them and recycle them
GTP = “switch”
What the properties of the ER translocon? Sec61 complex
- Sec61 protein has 2 parts → both sides of aqueous pore
- Inactive pore = plugged by part of protein
- When closed: plug + lateral gate closed
- Open: Signal peptide is in the lateral gate opening and translocating polypeptide is in the center (where the plug was)
- Lateral opening allows signal peptidase to have direct access to signal peptide to cleave it
- Active pore is open, but tightly sealed onto ribosome
- Inside of pore = neutral, polar
- 2 parts of pore open laterally to integrate TM helices into membrane
What are the steps of translocation of lumenal proteins from the cytosol to the ER lumen?
- Singal peptide triggers opening of the translocon
- Polypeptides are translocated in an extended, unfolded state right out of the ribosome → translocon → Mvt of polypeptide is driven by energy or translation pushing it out of the ribosome
- Signal peptidase (transmembrane protein) often removes signal peptide during translocation → not sequence-specific, but has preferred site
What are the steps for integration of TM helices in the membrane?
- Signal peptide starts translocation of lumenal part
- TM helix is recognized by translocon and integrated laterally into membrane during translation
- Cytosolic part is translated in the cytosol
What are Type 1 TM protein and Type 2 TM proteins?
Type 1 TM protein: N-terminus in lumen, C-terminus in cytosol
Type 2 TM protein: N-terminus in cytosol, C-terminus in lumen
What are the steps for integration of signal anchor in the membrane?
- Signal anchor open translocon like a signal peptide
- Translocon recognizes charged next to the signal anchor to determine orientation in membrane → positive changes in cytosol, negative charges in the lumen (will be neutralized that way)
- Signal anchor is recognized as a TM domain and integrated laterally
What post-translational modification do ALL proteins of the secretory pathway undergo?
N-linked Glycosylation → covalent bonding of glycans Asn side chain in Asn-X-Ser/Thr motif
- Help stabilize that native state
- Protect against proteases
- Function in cell surface signaling
*Multiple Asn side chains that have that motif can be motified this way
*Irreversible
What is a glycan?
Covalent attached to asparagine side chain → Asn-X-Ser/Thr motif
Structure starting from N-terminus of asparagine → 2x N-acetylglucosamine + multiple mannose + 3 glucose at the end
What is Oligosaccharyl transferase (OST)?
Enzyme responsible for N-Glycosylation, this PTM depends on OST accessibility
*It is a membrane protein
*Acts during translocation at the ER membrane
Can N-glycosylate multiple AsnxSer/Thre sites depending on accessibility
Do all TM protein have a signal peptide?
No, Type 2 only have anchor signal later in their sequence
What are the main differences in linkage for acylation and prenylation?
Acylation = bound to fatty acid (N-term or S from Cys bound to C=O from fatty acid)
N-Gly or N-Cys → amide linkage
Cys → thioesther linkage (REVERSIBLE)
Prenylation = bound to a fatty acid via prenyl group (Cys-CH2-fatty acid tail)
Cys → thioether linkage
How is the signal peptide released after being cleaved?
It’s released laterally by the translocon lateral gate
*same for signal anchor
