What are the 2 main functions of the protein coat in protein-coated vesicles?
- Concentrate the correct cargo (by binding specific receptor & adaptor proteins);
- Help to bend/curve the membrane during vesicle formation
What molecule is this?
What molecule does this become if phosphorylated at site 3, 4, and/or 5?
A phosphoinositide (PIP).
What is the function of BAR-domain proteins during vesicle formation?
To bend the donor membrane, which helps adaptor proteins to bind and shape the budding vesicle.
How does an adaptor protein act as a coincidence detector during vesicle formation?
It only allows further binding events if it is bound to both its specific membrane phosphoinostide and its specific transmembrane cargo receptor protein, so vesicles don't form unless the cell is signalled for export and cargo is ready to be transported.
How do BAR-domain proteins bend membranes?
The BAR domain is crescent-shaped and positively charged; electrostatic attraction between the BAR domain and the negatively charged phosphate groups of membrane phosphilipids causes the membrane to bend to conform to the shape of the protein.
What is the role of dynamin in clathrin-coated vesicle formation?
Once the vesicle is fully coated and connected only by a long "neck" to the donor membrane, dynamin helps to cleave the vesicle from the membrane.
What is the role of PIP phosphatases during vesicle transport?
They dephosphorylate the PIP of the vesicular membrane, which causes the adaptor proteins (and any remaining associated coat proteins) to dissociate, fully stripping the coat from the vesicle before it fuses with its target membrane.
What two monomeric GTPases regulate assembly and disassembly of COPI, COPII, and clathrin coats in protein-coated vesicles?
- Arf1 (COPI & clathrin)
- Sar1 (COPII)
What is the role of Hsp70 in vesicular transport?
It acts as a chaperone ATPase and helps to strip the protein coat from a vesicle before it fuses with its target membrane.
What is the role of AP2 in clathrin coat assembly during vesicle formation?
AP2 is an adaptor protein: it acts as a coincidence detector, binding both PI(4,5)P2 and cargo receptor proteins displaying endocytosis signals to initiate vesicle formation, and as a binding site for recruited coat proteins (clathrin).
What is happening here?
Vesicle formation for endocytosis at the plasma membrane: adaptor protein AP2 has bound 4 PI(4,5)P2 (1 at each of AP2's 4 subunits) and 2 transmembrane cargo receptors, and the simultaneous binding has caused the membrane to bend, which will help more AP2 proteins to bind.
What 2 domains of dynamin help it to cleave clathrin-coated vesicles from their donor membrane?
- PI(4,5)P2-binding domain (tethers dynamin to the membrane)
- GTPase domain (regulates the rate at which vesicles pinch off from the membrane)
What is the function of coat-recruitment GTPases?
To control the assembly and disassembly of clathrin coats on endosomes and of COPI & COPII coats on Golgi & ER membranes.
What is the role of ARF- and Sar1-GEFs in vesicular transport?
When a vesicle is ready to bud from a membrane, membrane-bound ARF- or Sar1-GAPs attract inactive ARF- or Sar1-GDP from the cytosol and bind them, causing them to release GDP to be replaced by GTP, activating the ARF or Sar1, which can then bind tightly to the membrane and recruit coat proteins for vesicle formation.
What happens when ARF-GDP or Sar1-GDP binds to the appropriate membrane-bound GEF during vesicle formation?
Binding causes the GTPase to release its bound GDP, which is quickly replaced by GTP from the cytosol, triggering a conformational change: the GTPase exposes an amphipathic α-helix that integrates into the membrane, tightly binding the GTPase to the membrane and allowing it to recruit coat proteins for vesicle formation.
In protein-coated vesicles, what conformational change is triggered by GTP hydrolysis of coat-recruitment GTPases?
Hydrolysis of bound GTP to GDP causes the GTPase's amphiphilic α-helix to pop out of the vesicular membrane, causing the GTPase to dissociate from the membrane and the protein coat to disassemble.
Of clathrin-, COPI-, and COPII-coated vesicles, which shed their protein coats immediately after pinching off from their donor membrane?
Clathrin- and COPI-coated vesicles; COPII coats are stable enough to stay sealed around the vesicle even after the coat-recruitment GTPases have dissociated, so coat disassembly is only complete when kinases at the target membrane phosphorylate the coat proteins to prepare the vesicle for fusion.
What triggers coat disassembly in COPI-coated vesicles?
The curvature of the vesicle membrane as it begins to pinch off from the donor membrane: ARF-GAP recruited to the COPI coat during assembly senses the increase in lipid packing density and becomes activated, and the active ARF-GAP inactivates ARF (the coat-recruitment protein), causing the coat to disassemble.
Why do COPII-coated vesicles keep their protein coats until they reach their target membrane?
Once the vesicle has budded off, the sealed COPII coat is stabilized by many cooperative interactions (including with the cargo receptors in the vesicular membrane), so it stays intact until the coat proteins are phosphorylated by kinases at the target membrane docking site.
What two structural features make the plasma membrane relatively stiff and flat compared to other membranes in the cell?
- Cholesterol-rich lipid composition
- Underlying actin-rich cortex
What are Sec23, Sec24, Sec13, and Sec31?
COPII coat proteins involved in COPII-coated vesicle formation: Sec23/24 form the inner layer of the coat, and Sec13/31 form the outer layer.
What regulatory mechanism follows from the proposal that vesicle coat-recruitment GTPases have a built-in hydrolysis "timer"?
- Coat-recruitment GTPases hydrolyze their own bound GTP at a slow, predictable rate
- Vesicle formation is only successful if assembly is faster than hydrolysis; otherwise, it disassembles before it's finished
- Conditions must be ideal for vesicle formation in order for assembly to outpace hydrolysis
Why do clathrin coats need to deform the donor membrane during vesicle formation (as opposed to just capturing cargo proteins)?
Clathrin-coated vesicles bud from the plasma membrane, which is stiffer and flatter than organelle membranes, so extra force is required to induce curvature and allow budding and pinching off of vesicles.
Why is the primary function of COPI and COPII coat proteins only to capture appropriate cargo proteins (vs. clathrin coat proteins, which also need to deform the membrane)?
COPI- and COPII-coated vesicles bud from regions of intracellular membranes where the membrane is already curved, so they don't require much extra force to bend the membrane during vesicle budding and pinching-off.
What surface molecules on transport vesicles and their target membranes ensure specificity of vesicle traffic?
- Transport vesicles display surface markers that ID them according to origin and cargo type
- Target membranes display complementary receptors that recognize appropriate vesicle markers
What two protein systems ensure specificity of targeting during vesicular transport?
- Rab proteins & Rab effectors (direct vesicles to specific points on target membranes)
- SNARE proteins & SNARE regulators (mediate fusion of vesicle and target membrane lipid bilayers)
What is Rab-GDP dissociation inhibitor (GDI)?
A protein that binds inactive Rab-GDP in the cytosol, keeping it soluble until it is activated by membrane-bound Rab-GEFs.
What happens when a Rab protein is activated by a membrane-bound Rab-GEF during vesicular transport?
Activation triggers a conformational change that exposes a lipid anchor in Rab, which tightly binds it to the membrane, and the GTP- and membrane-bound Rab is able to recruit and bind Rab effectors.
What is the role of Rab effectors during vesicle transport?
Once recruited and bound by active, membrane-bound Rab proteins, they act as downstream mediators of vesicle transport, membrane tethering, and membrane fusion.
During vesicle transport, how is the concentration of active Rab (and Rab effectors) determined at vesicle and/or target membranes?
By the rate of GTP hydrolysis at the GTP-bound Rab proteins (which inactivates Rab).
During vesicle transport, what is the function of Rab effectors that are motor proteins?
When activated by vesicular membrane-bound Rab proteins, the effectors propel vesicles along actin filaments or microtubules to their target membrane.
During vesicle formation, what is the function of Rab effectors that are tethering proteins?
Once bound by target or vesicular membrane-bound Rab proteins, the effectors extend as long threads or form large protein complexes to link two membranes together and facilitate membrane fusion.
How do Rab proteins confer an additional level of specificity on labelling membranes for vesicle transport?
Every organelle has a different Rab protein associated with it, so interaction occurs only between membranes that have the right combination of Rab proteins.
How does the Rab cascade in endosome maturation (Rab5 domains replaced by Rab7 domains) result in a difference of function between early (Rab5) and late (Rab7) endosomes?
Rab5 and Rab7 recruit different Rab effectors, so the entire compartment is reprogrammed:
- Alters membrane dynamics (including incoming/outgoing traffic)
- Repositions the endosome to face away from the plasma membrane toward the cell interior
How does the self-amplifying nature of Rab domains affect the directionality of endosome maturation?
- Early endosomes have Rab5 domains, and late endosomes have Rab7 domains
- Rab5 effectors include Rab7-GEF; Rab7 effectors include Rab5-GAP
- Rab5 activity adds Rab7, and Rab7 activity removes Rab5: Rab5 is unidirectionally and irreversibly replaced by Rab7
- Early endosomes unidirectionally and irreversibly become late endosomes
- Early endosomes unidirectionally and irreversibly become late endosomes
Why is there an energy barrier involved in membrane fusion during vesicle transport?
2 lipid bilayers won't fuse until they are within 1.5 nm of each other, and they can't get that close until water molecules are displaced from the hydrophilic surface of the membrane, which is an energetically unfavourable process.
What is the role of SNARE proteins in vesicle transport?
To help overcome the energy barrier involved in displacing water molecules between membranes, catalyzing fusion of vesicle and target membranes.
How do SNARE proteins work to catalyze membrane fusion during vesicle transport?
Complementary v-SNAREs and t-SNAREs on vesicle and target membranes come together to form trans-SNARE complexes, using energy freed when their interacting helical domains wrap around each other to simultaneously pull the membrane faces together and squeeze out water molecules from the interface.
What is the main structural difference between t-SNAREs and v-SNAREs?
A v-SNARE is a single polypeptide chain, while a t-SNARE is usually made up of 3 proteins (so a trans-SNARE complex has a total of 4 helical domains, all bundled together).
How do SNARE proteins provide an additional layer of specificity in the vesicle transport process?
Different membrane types have different SNAREs, and v- and t-SNARE pairing is highly specific, so only certain combinations of v- and t-SNAREs will result in successful interaction and membrane fusion.
True or false: In the cell, complementary v- and t-SNAREs initiate rapid fusion of vesicle and target membranes without any assistance from other proteins.
False: liposomes containing only purified SNAREs will fuse very slowly in vitro; in the cell, several proteins are recruited to the fusion site to help SNAREs accelerate fusion.
How can Rab proteins regulate the availability of SNARE proteins during vesicle transport?
t-SNAREs in target membranes are often associated with inhibitory proteins; Rab proteins and effectors can trigger the release of SNARE inhibitory proteins so that SNARE proteins are concentrated and activated in the right location on the membrane so that incoming vesicles can bind and fuse with the membrane.
Where are t-SNARE and v-SNARE proteins usually found in the cell?
- t-SNAREs: target membranes
- v-SNAREs: vesicle membranes
(Note: Sometimes t-SNAREs are in vesicle membranes and v-SNAREs are in target membranes--all that matters is that v-SNAREs pair with t-SNAREs.)
What is the role of NSF in vesicle transport?
NSF cycles between membranes and the cytosol and catalyzes the disassembly of stable trans-SNARE complexes where vesicle and target membranes have fused, freeing v- and t-SNAREs for new fusion events.
Why is it important that trans-SNARE complexes must be actively disassembled by NSF in order to reactivate the associated SNARE proteins?
NSF-mediated disassembly prevents membranes from fusing indiscriminately: if the t-SNAREs in a target membrane were always active, they could fuse with any passing membrane containing the right v-SNARE whenever the two membranes made contact, not just when specific transport events were activated.
How does the tetanus toxin interact with SNARE proteins to cause muscles to lock up?
The toxin cleaves SNARE proteins in nerve terminals, so vesicles carrying neurotransmitters are no longer able to fuse with the synaptic membranes, and synaptic transmission controlling muscle contraction and relaxation is blocked.
What is KDEL?
A common C-terminal signal in ER resident proteins; receptors in the Golgi and the vesicular tubular cluster recognize and bind KDEL and trigger vesicle formation to send the protein back to the ER.
How are the cis and trans faces of the Golgi oriented within the cell?
- Cis face: facing ER (receives incoming vesicles)
- Trans face: facing away from ER (buds off secretory vesicles)
What is the difference between plant and animal cells with respect to organization of the Golgi?
- Animal cells: 1 large Golgi complex near the nucleus
- Plant cells: 1000s of smaller Golgi bodies, scattered throughout the cell
What kind of sugars are initially attached to proteins in the ER?
14-oligosaccharides (later modified in the Golgi).
True or false: Each compartment of the Golgi complex contains the same enzymes.
False: each compartment contains different enzymes and modifies proteins in different ways.
What are the two main models proposed to explain how the Golgi works?
- Cisternal maturation: each enclosed compartment evolves from accumulated vesicular clusters
- Vesicular transport: compartments are stable and stationary, and vesicles just deliver materials for protein modification
(Reality is probably somewhere in between?)
How are high-mannose oligosaccharides completed in the Golgi?
Sugars are cut off, but nothing new is added.
How are complex oligosaccharides completed in the Golgi?
Some sugars are cut off, and some sugars are added.
How does oligosaccharide accessibility determine how a protein's sugar group will be modified in the Golgi?
- Less accessible: sugars can be cut off, but not added, resulting in a high-mannose oligosaccharide
- More accessible: sugars can be cut off and added, resulting in a complex oligosaccharide
Why is it important that different types of cells have different types of glycosyl transferases?
Different cells make different proteins with different sugars attached; since sugars are only ever found on the extracellular side of a cell's plasma membrane, this means that every distinct cell type expresses a distinct sugar tag specific to that cell type.
What type of ATP pump maintains the low pH of lysosomes?
V-type ATPases: proton pumps that hydrolyze ATP to fuel active transport of H+ into the lysosome.
What are acidic hydrolases?
A set of enzymes found in lysosomes that are able to digest all types of macromolecules (but are only functional at low pH).
Once synthesized, how are acidic hydrolases transported to lysosomes?
They are transported as inactive precursors and only become functional after they are cleaved in the lysosome.
How do lysosomes protect themselves from being digested by their own enzymes?
Lysosomal membrane proteins are very heavily glycosylated on the inner face of the membrane, which keeps digestive enzymes from getting close enough to the actual proteins and lipids to digest the membrane.
What structures in plant cells are equivalent to animal lysosomes?
True or false: All eukaryotic cells are capable of phagocytosis.
False: unicellular organisms are capable of phagocytosis (it's how they eat), but in multicellular organisms, only certain specialized cells are capable of phagocytosis.
What is the role of Rho-GTPase in phagocytosis?
- Active Rho-GTPase activates local PI kinases
- Active PI kinases phosphorylate nearby PIs to PI(4,5)P2
- Nearby actin binds PI(4,5)P2 (actin rearrangement)
- Actin rearrangement results in formation of pseudopods to engulf particles
What is the role of PI 3-kinase in phagocytosis?
Once the pseudopods have enclosed
What 2 resident ER proteins involved in glycosylation bind glucose on glycosylated proteins to keep them within the ER until they are folded correctly?
- Calnexin (membrane-bound)
- Calreticulin (soluble)
When does ubiquitin ligase attach a poly-ubiquitin chain to proteins in the ER?
When the protein is misfolded and needs to be sent to a proteosome for degradation (retrotranslocation).
What is the difference between glycosyl transferase and glucosyl transferase?
- Glycosyl transferase adds specific sugar groups to proteins that are being modified in the Golgi
- Glucosyl transferase adds glucose to misfolded proteins in the ER so that they are bound by calnexin or calreticulin while an attempt is made to refold them
What is the function of recycling transport vesicles?
To return membrane-bound receptors back to the plasma membrane after their cargo has been delivered within the cell.
What 3 domains are characteristic of nuclear receptor proteins?
- Ligand-binding domain (binds incoming signal)
- DNA-binding domain (binds region of target DNA)
- Transcription-activating domain (binds transcription factors to upregulate target gene expression)
What activates PKA and PKC?
- PKA: cAMP
- PKC: Ca2+
What are the 3 statements made by the Cell Doctrine (1838)?
- Cells are the smallest living unit: all organisms are made of 1 or more cells;
- Cells are distinct units with specific tasks;
- A cell can only come from another cell by cell division
What are the 4 basic structures found in all cells?
- Plasma membrane
What are the 3 most highly conserved gene families among all domains of life?
- Translation genes
- Amino acid transport & metabolism genes
- Coenzyme transport & metabolism genes
What 2 characteristics are found in some archaea that are not found in bacteria or eukaryotes?
- Membrane lipids with branched hydrocarbons
- Growth at >100 ºC
What 2 structures are found in animal cells that are absent in plant cells?
- Centrosomes (with centrioles)
What 4 structures are found in plants cells that are absent in animal cells?
- Cell wall
- Central vacuole
What three components are found in greater concentrations within lipid rafts than within the rest of the plasma membrane?
- GPI-anchored proteins
Why is Na+/glucose cotransport paired with a Na+-K+ pump in gut epithelial cells?
Active transport of glucose relies on passive diffusion of Na+ into the epithelial cell, so Na+-K+ pumps are needed to pump Na+ back out of the cell to maintain the concentration gradient driving glucose symport.
What type of pump is the Ca2+ pump in the sarcoplasmic reticulum of muscle cells?
A P-type ATPase.
What is the basic structure of a phosphoglyceride?
- 2 fatty acid tails ester-linked to a 3-C glycerol backbone
- Phosphate group attached to the 3rd C of glycerol
- 1 of several possible head groups attached to the phosphate group
What are the 3 most abundant phosphoglycerides in mammalian cell membranes?
What are the 2 main basic structural differences between phosphoglycerides and sphingolipids?
- Phosphoglycerides are based on a glycerol backbone, while sphingolipids are based on a sphingosine backbone
- Phosphoglycerides have 2 fatty acid tails; sphingolipids have 1 fatty acid tail next to the fatty chain that is part of sphingosine
What is the most common sphingolipid?
What do phosphatidylcholine and sphingomyelin have in common?
Both have head groups composed of choline attached to a phosphate group.
How do high concentrations of cholesterol in eukaryotic plasma membranes maintain the fluidity of membranes?
Cholesterol prevents the hydrocarbon chains of membrane lipids from coming together and crystallizing.
How does cholesterol make lipid bilayers less permeable?
The steroid rings interact with and partly immobilize the first few CH2 groups of phospholipid tails in the membrane, and the reduced mobility of lipids near the surface of the membrane makes it harder for molecules to squeeze between them.
Where is phosphatidylserine concentrated in living cells?
On the cytosolic face of the plasma membrane.
Which enzyme, involved in cell signalling, requires phosphatidylserine for its activity?
Protein kinase C (PKC).
What membrane phospholipid does protein kinase C (PKC) require for activity during cell signalling?
What are phospholipases?
Enzymes within the plasma membrane that are activated by extracellula signals to cleave specific membrane phospholipids, generating fragments of these molecules that act as short-lived intracellular mediators.
What happens to phosphatidylserine when a cell undergoes apoptosis?
It flips from the cytosolic to the extracellular face of the plasma membrane, signalling neighbouring cells to phagocytize and digest the dead cell.
What two interactions lead glycolipids to preferentially self-associate into lipid raft phases?
- Hydrogen bonds between their sugar head groups
- van der Waals forces between their long, straight hydrocarbon chains
What is the main structural difference between sphingolipids and glycolipids?
- Sphingolipids: sphingosine is attached to a phosphate group, which is attached to a head group
- Glycolipids: sphingosine is directly attached to a sugar group (no phosphate)
How are glycolipids distributed in cell membranes?
Whether in the plasma membrane or in intracellular membranes, they are found exclusively in the monolayer facing away from the cytosol.
What gives gangliosides a net negative charge?
One or more sialic acid (NANA) moieties within their sugar head groups.
What class of membrane lipids do gangliosides belong to?
Glycolipids (sphingosine backbone, sugar head group).
Where are gangliosides most abundant?
In the plasma membranes of nerve cells.
What are lectins?
Proteins that bind to carbohydrates.
How do lectins contribute to cell recognition and cell-cell adhesion events?
By binding sugar groups on glycolipids and glycoproteins on the outer face of cell membranes.
Why might it be beneficial for gangliosides to be concentrated in nerve cell plasma membranes?
Their negative charge alters the electrical field across the membrane and the concentrations of ions (especially Ca2+) at the membrane surface.
What is one possible function of glycolipids in the exposed apical surface of epithelial cell plasma membranes?
The sugar groups may protect the membrane from harsh conditions at the apical surface (e.g. low pH, high concentration of degrading enzymes).
Which membrane lipid acts as a cell-surface receptor for the cholera toxin?
How does the cholera toxin cause diarrhea?
- Toxin binds and enters intestinal epithelial cells displaying ganglioside GM1 on their surface
- Entry into cell leads to prolonged increase in intracellular [cAMP]
- Increased [cAMP] leads to large efflux of Cl-
- Cl- efflux leads to secretion of Na+, K+, HCO3-, and water into the intestine
How are membrane proteins that are fully exposed at the external cell surface attached to the membrane?
By a covalent linkage to a lipid anchor in the outer monolayer of the plasma membrane, via a specific oligosaccharide.
What is the main difference between membrane proteins and membrane-associated proteins?
Unlike membrane proteins, membrane-associated proteins don't extend into the hydrophobic interior of the lipid bilayer at all, and are instead bound to either face of the membrane by noncovalent interactions with other membrane proteins.
Why are α-helices and β-barrels found in the membrane-spanning domains of transmembrane proteins?
Peptide bonds are polar and the interior of the bilayer is nonpolar, so the peptide bonds are driven to form hydrogen bonds with each other; hydrogen-bonding between peptide bonds is maximized if polypeptides form regular α-helices or arrange into β-barrels as they cross the bilayer.
What are the two main classes of membrane proteins that mediate transmembrane transport of small molecules?
What is the functional consequence of cell using transporters and channels to generate inorganic ion-concentration differences across their lipid bilayers?
The difference creates an electrochemical gradient, allowing cell membranes to act as a store of potential energy to drive various energy-requiring processes in the cell.
What is the main factor that determines the rate at which a molecule will diffuse through a lipid bilayer (ignoring membrane proteins)?
The molecule's relative hydrophobicity: the more hydrophobic (or nonpolar) it is, the more easily it will diffuse across a lipid bilayer.
What type of molecules diffuse most readily across a lipid bilayer?
Small, nonpolar molecules (e.g. CO2, O2).
What 2 factors keep ions from diffusing across lipid membranes without help from proteins?
- High degree of hydration
What 2 factors combine to create a membrane potential across a cell membrane?
- Concentration gradient of a particular solute
- Electrical gradient (the difference in electrical potential on either side of the membrane)
True or false: A transporter may expose its solute-binding site on one side of the membrane or other, but never on both sides at the same time.
True: there is an intermediate state in between, where the solute is inaccessible from either side of the membrane.
What does the Vmax value of a transporter measure?
The maximal rate of transport, i.e. the rate at which the transporter can flip between its conformational states (open on one side of the membrane or the other), reached when all solute-binding sites are occupied.
What is the difference between competitive and noncompetitive inhibitors?
- Competitive inhibitors compete for the same binding site on an enzyme or transporter as the solute
- Noncompetitive inhibitors bind elsewhere on the enzyme or transporter and alter its structure
What does the Km value for a transporter measure?
The concentration of solute where the transport rate is at half of its maximum value (Vmax), reflecting the characteristic affinity of the transporter for its solute.
What are the 3 main mechanisms of active transport found in cells?
- Coupled transporters
- ATP-driven pumps
- Light- or redox-driven pumps (only found in prokaryotes, mitochondria, and chloroplasts)
What is the functional significance of the pseudosymmetry of transporters?
Like enzymes, transporters can work in the reverse direction if ion and solute gradients are reversed, so having two halves of the transporter inverted relative to each other allows alternating access to the ion- and solute-binding sites in the centre of the transporter.
In coupled transport, which ion is the most common co-transported ion in animal cell plasma membranes?
Which transporter in the plasma membrane maintains the Na+ gradient by pumping out Na+ that enters the cell during coupled transport?
An ATP-driven Na+-K+ pump.
What are the 3 types of filaments in the cytoskeleton?
- Actin filaments
- Intermediate filaments
What three properties of cells are determined by the activity and structure of the cytoskeleton?
What are the 3 main functions of actin filaments?
- Shape of the cell's surface
- Whole-cell locomotion
- Pinching of 1 cell into 2 during cell division
What are the 3 main functions of microtubules?
- Position membrane-enclosed organelles
- Direct intracellular transport
- Form mitotic spindle to segregate chromosomes during cell division
What is the main function of intermediate filaments?
To provide the cell with mechanical strength.
True or false: Intermediate filaments are found in the cytoskeleton of all eukaryotic cells.
False: intermediate filaments are only found in vertebrates, nematodes, and molluscs (soft-bodied animals).
What type of protein acts as a GAP to inactivate G proteins in eukaryotic cells?
RGS (regulator of G protein signalling).
What acts as a GEF for G proteins?
Activated GPCRs (G-protein-coupled receptors).
What happens when a G protein is activated by its associated GPCR?
- Gα subunit releases its bound GDP, and GTP binds in its place
- GTP binding triggers conformational change: G protein dissociates from GPCR, and Gα subunit dissociates from Gβγ subunit pair
- Gα and Gβγ go on to activate other signalling proteins
Which subunit of a G protein is a GTPase?
How are most enzyme-coupled receptors activated?
Ligand-binding promotes the dimerization of two enzymatic receptors or of a receptor and its coupled enzyme, and the interaction of the two subunits results in the activation of the cytoplasmic catalytic domain.
How does adrenaline (epinephrine) stimulate glycogen breakdown in skeletal muscle cells?
- Adrenaline binds to a GPCR
- Active GPCR increases intracellular [cAMP]
- cAMP activates an enzyme that promotes glycogen breakdown and inhibits an enzyme that promotes glycogen synthesis
In the context of cell signalling, what happens in desensitization?
Prolonged exposure to a stimulus decreases the cell's response to that level of stimulus, so the cell is able to detect the same percentage of change in the signal over a wide range of stimulus strengths.
What is the underlying mechanism behind desensitization of cells to certain stimuli?
Negative feedback operating with a short delay:
- Strong response modifies the signalling machinery so that it resets itself to become less responsive to the same level of signal
- Delay means a sudden increase in signal can stimulate the cell again for a short time before negative feedback has time to kick in
What three senses rely on GPCRs?
What are RGS proteins?
Regulators of G-protein signalling: they act as GAPs on specific sets of G-protein α-subunits to shut off G-protein-mediated responses in all eukaryotes.
What is adenylyl cyclase?
A large, multipass transmembrane protein, usually regulated by G proteins and Ca2+, that converts ATP to cyclic AMP.
What enzyme catalyzes the formation of cAMP from ATP?
What enzyme catalyzes the destruction of cyclic AMP?
Cyclic AMP phosphodiesterase.
What happens during signalling involving extracellular signals that increase [cAMP] in target cells?
- Signals activate GPCRs coupled to a stimulatory G protein (Gs)
- Activated α subunit of Gs binds & activates adenylyl cyclase
- Active adenylyl cyclase synthesizes cAMP from ATP
What happens during signalling involving extracellular signals that decrease [cAMP] in target cells?
- Extracellular signals activate GCPRs coupled to an inhibitory G protein (Gi)
- Activated α subunit of Gi binds & inhibits adenylyl cyclase, which inhibits cAMP synthesis
How does cAMP trigger cell responses in most animal cells?
By activating PKAs (cAMP-dependent protein kinases), which phosphorylate specific Ser or Thr residues on selected target proteins to regulate their activity.
Why can the effects of cAMP vary widely between different cell types?
PKAs (cAMP-dependent protein kinases) in different cell types target different proteins for phosphorylation, resulting in different effects of increased or decreased [cAMP].
What subunits are present in an inactive PKA (cAMP-dependent protein kinase)?
2 catalytic subunits and 2 regulatory subunits, all bound together in a complex.
What happens when cAMP binds to an inactive PKA?
- cAMP binds the regulatory subunits of PKA
- Conformational change causes regulatory subunits to dissociate from the PKA complex
- Catalytic subunits are released and activated to phosphorylate specific target proteins
How do the regulatory subunits of PKA help to localize the kinase within the cell?
Specialized anchoring proteins (AKAPs) bind to both the regulatory subunits and to a component of the cytoskeleton or an organelle membrane, tethering the PKA complex to a particular subcellular compartment.
What are AKAPs?
A-kinase (PKA) anchoring proteins: specialized proteins with binding sites for the regulatory subunits of PKA and for specific components of the cytoskeleton or of the membane of an organelle that tether PKA and localize it to a particular region of the cell.
Where are actin filaments most highly concentrated?
In the cortex, just beneath the plasma membrane.
What homologue of tubulin in prokaryotes is involved in cell division?
What is FtsZ?
A tubulin homologue involved in cell division in prokaryotes.
Which two homologues of actin contribute to cell shape in bacteria?
What are MreB and Mbl?
Homologues of actin found in bacterial cells, where they contribute to cell shape by acting as a scaffold to direct cell wall synthesis.
What is ParM?
A bacterial actin homologue encoded on certain plasmids that assembles into spindles to push replicated plasmids apart during plasmid replication (similar to a mitotic spindle).
What is TubZ?
A distant relative of tubulin and FtsZ that helps to push replicated plasmids apart during plasmid replication in some bacterial cells.
What are the 3 isoforms of actin found in vertebrates?
Which isoform of actin is only found in vertebrate muscle cells?
What are phalloidins?
Toxins isolated from the Amanita mushroom that bind tightly along the sides of actin filaments and prevent their depolymerization,
What is α-actinin?
A dimeric protein that stabilizes contractile bundles by acting as a spacer between actin filaments, making room for myosin-II between the actin and α-actinin binding sites.
Which type of cytoskeletal filament forms the contractile ring during eukaryotic cell division?
What are the 2 main functions of actin filaments?
- Cell shape
- Whole-cell locomotion
Which group of proteins promotes the assembly of branching actin filaments?
ARPs (actin-related proteins).
How does an ARP complex promote the growth of branching actin filaments?
- Activating factor binds & activates ARP complex
- Conformational change allows binding between active ARP complex and free actin monomers
- ARP complex binds to some point along a growing actin filament and recruits monomers to build a filament branching off of the existing filament
What is the difference between active ARP complexes and active formins with respect to motility during actin filament nucleation?
- ARP complexes stay anchored at the minus end of the growing filament, capping the minus end and attaching the new filament to the preexisting one
- Formins move toward the plus end of the growing filament after each addition of monomers so that they stay at the growing plus end while active
What is CapZ?
A capping protein that binds to the plus ends of actin filaments to prevent polymerization (addition of free actin monomers) at that end, allowing growth at the minus end only.
Which capping protein binds to the plus ends of actin filaments?
Which capping proteins bind to the minus ends of actin filaments?
Arp2/Arp3 (ARP complex) and tropomodulin.
What is a contractile bundle?
An actin array containing straight filaments organized in loose bundles and stabilized by α-actinin.
What is filamin?
A dimeric protein that stabilizes actin arrays organized in net-like structures by strengthening the associations between overlapping filaments.
What is fimbrin?
A protein that stabilizes tight parallel actin bundles by packing the filaments together, preventing motor proteins from incorporating into the bundle.
What are formins?
A family of dimeric proteins that regulate nucleation of straight (unbranched) actin filaments.
What is the main function of microtubules?
To direct organelle and vesicle traffic within the cytoplasm.
What are myosins?
A large family of dimeric motor proteins that interact with actin filaments by "walking" along the filament toward the plus end (except for 1 that walks toward the minus end).
What are the 5 main domains of myosins?
- N-terminus (head)
- Myosin light chains
- Neck/hinge region
- Long α-helix (~2000 AAs; coiled-coil structure in dimer form)
Why does rigor mortis set in after death?
Myosins stay tightly bound to the actin filaments in muscle cells until they bind ATP: since the body is no longer producing ATP, there is nothing to release myosin from actin, and so the whole structure stays rigid.
What is the main role of myosin light chains?
To promote hydrolysis of ATP on the head groups of active myosin, which prompts the whole motor protein to bind again to an actin filament.
What is persistence length?
The minimum length that a cytoskeletal filament must reach before it is long enough to bend.
What is the persistence length of actin filaments?
20 to 40 μm (very flexible).
What is the persistence length of microtubules?
In the mm range (very stiff).
What is profilin?
A protein that binds free actin monomers to promote rapid growth at the plus ends of actin filaments.
What is thymosin?
A protein that binds free actin monomers to prevent their addition to actin filaments, inhibiting polymerization and growth of actin filaments.
True or false: A free actin monomer may have profilin or thymosin bound to it, but never both at the same time.
True or false: Actin and tubulin have inherent ATP-/GTP-ase activity, allowing them to hydrolyze their own bound ATP/GTP to ADP/GDP over time.
True (and this drives the conversion of T-form to D-form subunits within filaments).
What is tropomodulin?
A capping protein that binds at the minus ends of actin filaments to prevent depolymerization; stable over longer periods of time than Arp2/Arp3, so is favoured in long-lived filaments (e.g. in muscle tissue).