Cell Bio Midterm #2 Flashcards
lec 11-
what happens in protein targeting
messenger RNA (mRNA) is transcribed in the nucleus, then transported to the cytoplasm where it is translated by ribosomes to specific proteins.
What are the two mechanisms of protein targeting
signal-based targeting and vesicle-based targeting
what is signal-based targeting used by
proteins that are destined to the ER, mitochondria, nucleus, chloroplasts, and peroxiosomes
what is vesicle-based targeting used by
proteins that are excreted into the extracellular space, inserted into plasms membrane, and by proteins that are destined for the Golgi or lysosomes
what is signal-based targeting encoded and directed by
specific amino acid sequences in the protein
what do the specific amino acid proteins in signal-based targeting serve as
adaptors for other proteins and protein complexes that are responsible for moving protiens arounf the cell
the nucleus contains two membranes that make up the what
nuclear envelope
what does Tom stand for
translocon of outer membrane
what does the N-terminal signal sequence bind to
the Tom protein complex on the outer mitochondrial membrane
What does the N-terminal and Tom complex contain
a receptor that recognizes the signal sequence and a channel that allows the protein to pass through the outer membrane
what happens in Vesicle-based trafficking
proteins are transported from the endoplasmic reticulum (ER) lumen through the golgi apparatus to other membrane compartments
what is the golgi apparatus responsible for
transporting, modifying, and packaging proteins and lipids into vesicles for delivery to targeted destinations
What types of proteins use the vesicle-based trafficking mechanism to reach their final destination
- proteins that are secreted from the cell via exocytosis
- plasma membrane porteins
- lysosomal proteins
- golgi proteins
what is budding
the formation of a vessicle from an organelle, such as the ER of Golgi
what is budding facilitated by
coat proteins and GTP-binding proteins that bind to the cytosolic face of the organelle membrane
what do coat proteins do
generate curvature in the membrane, help determine which proteins will be loaded into the vesicle that is forming, and ensure that v-SNAREs are present
what do proteins such as dynamin do
associate with the neck region of the forming vesicle and pinch the vesicle off of the donor membrane
what energy source does dynamin require to associate with the neck region of the forming vesicle
energy in the form of GTP
once the vesicle has formed what happens
the coat proteins are shed
what causes the relase of the coat proteins
GTP-binding proteins hydrolyzing GTP to GDP
what is the benefit of v-SNAREs interacting with t-SNAREs
The vesicle membrane is brought into close proximity to the target membrane
what are the 3 classes of vesicles
1) COPII vesicles
2) COPI vesicles
3) clathrin vesicles
what class of vesicles are used during endocytosis
clathrin vesicles
what do CopII vesicles do
move cargo from the ER to the cis-Golgi
what do COPI vesicles do
move cargo between Golgi compartments and from cis-Golgi to ER
what do Clathrin vesicles do
move dysfunctional proteins from trans-Golgi to lysosomes for degradation
what is endocytosis
a process where cells can engulf material from the extracellular matrix
how are endocytic vesicles foemed during endocytosis
the plasma membrane surrounds the material to be engulfed and fuses to form endocytic vesicles
what is the purpose of endocytosis
to internalize material from the extracellular environment
how is degradation achieved
by lysosomal enzymes and acidic pH created by V-class proton pumps
why do plasma membranes need to be degraded
so they can be endocytose
what is the endosome membrane used for
used to form internal vesicles which are then releases into the lysosome for degradation
what is the purpose of autophagy
to degrade and therby recycle old organelles and protiens found in the cytosol
what are the 4 general steps
- Double-cup membrane structure formed
- This autophagosome surrounds material to be degraded
- The autophagosome fuses with the lysosome
- Lysosomal enzymes degrade material, allowing for the recycling of cellular components
when is autophagy often initiated
during periods of nutrient deprivation
how does autophagy begin
with the formation of an autophagosome (double-membrane cup-shaped structure) that envelops the material to be degraded
what does a autophagosome fuse with and what happens once it fuses
fuses with a lysosome
contents are then degraded and recycled
in multicellular organisms what is cell signalling required for
- organismal development
- organization into tissues
- co-ordination of activities
- control od growth and division
in single-cell roganisms what is cell signalling required for
adaption to changes in the environment
what is signal transduction
conversion of an extracellular signal into a cellular response
why do we need signal transduction
because cells must be able to change aspects of their structure in response to signals in their environment
what are the 4 common types of signals
- chemical
- physical
- endogenous
- exogenous
what are endogenous signals
signals that can be generated internally
what are endogenous signals divided into
endocrine, paracrine, autocrine, and plasma membrane-attached protein signals
what are exogenous signals
signals that can be generated from the outside of the organism
what do exogenous signals include
photons of light, odors, medications, invading micro-organisms
what are endocrine signallers calles and what do they do
- endocrine signaling molecules are called hormones (e.g., insulin, epinephrine)
- released into bloodstream to access distant target cells
what happens in paracrine signaling
- a signal is released by a nearby cell onto a target cell
- can create a gradient of signalling molecules
- e.g., neurotransmitters, growth factors
what is an autocrine signal
a signal that acts on the cell that released it
- e.g., cytokines, certain growht factors
what are plasma membrane-attached protein signalling
- signaling molecules embedded in the plasma membrane of one cell to activate receptors on another cell following direct contact
- these molecules can also be cleaved from the membane and become soluble signals
- e.g, adhesion molecules on immune cells
why do we need plasma membrane receptors
- receptors provide exquisite selectivity for particular signals
-They allow for graded responses depending upon the amount of signal present - They allow a single signal to produce different responses in different cell types
- They provide signal amplification
- Most signals are not able to cross the plasma so they detect these signals and initiate intracellular responses
what bonds to the receptor
ligand or agonist
what does receptor activation really mean
activation refers to a change in the conformation of the receptor that signals the presence of the ligand
what is the only way for a cell to respond to a signal
if it possesses a receptor for that particualr signal
what are receptors
proteins that physically interact with the signal they are sensitive to
what is a ligand
a molecule that can tightly bind to a receptor and activate it (aka receptor agonist)
how is an intracellular transduction pathway activated
when a ligand binds to its receptor, the receptor undergoes a confomational change
what can a conformational change do (other than activating an intracellular transduction pathway)
- turn on enzymatic activity off the receptor
- open pore in receptor for ions to flow through
- activate a kinase bound to the receptor
- activates a G-protein
what are plasma membrane receptors used to detect
signals that cannot cross the plasma membrane
what are the three regions that plasma membrane receptors consist of
- extracellular domain
- transmembrane domain
- intracellular (cytoplasmic) domain
what does the extracellular domain possess
a specific binding site for signal
where is the transmembrane domain
embedded in the plasma membrane
what is the intracellular comain associated with
enzymes or other proteins that can help transduce signal
what are the three main classes of plasma membrane receptors
G-protein coupled receptors
- ligand-gated ion channels
- enzyme-linked receptors
what happens with G-protein coupled receptors
- Receptor interacts with heterotrimeric G-protein
- Ligand binding causes G-protein to dissociate from receptor
- Activated G-proteins interact with intracellular targets to regulate cell function
what happens with ligand-gated ion channel
- Ligand binds to receptor, causing opening of pore in the receptor
- Ions flow freely through the pore
what happens with enzyme-linked receptors
- Receptor either has intrinsic enzyme activity or associates with an enzyme
- Ligand binds to receptor, causing activation of enzyme
what do intracellular receptors detect
signalling molecules that have crossed the plasma membrane
(e.g., steroids and thyroid hormones)
what do intracellular receptors regulate
gene expression
ligands bind to the binding site of the receptor using what type of bond
non-covalent interactions
what does affinity of ligand refer to
the strength of interaction between ligand and receptor
what does specificity refer to
the ability of receptor to discriminate between ligands
what does binding of molecules to allosteric site modify
degree of receptor activation by ligand
what are the 3 factors that affect the magnitude of cellular response to receptor activation
1) amount of signaling molecule present: response increases with increases concentration
2) affinity of receptor for signaling molecule: higher affinity results in a response at a lower dosage
3) receptor expression: maximal response increases with increasing number os receptors
what do antagonists do
inhibit receptor activation by agonists.
- reversible or irreversible
what are the two types of antagonist
competitive: competes with agonist for binding site
non-competitive: binds to allosteris site on receptor
how does a competitive agonist influence cells
increases agonist concentration required to achieve desired effect
how does non-competitive agonist influence cells
either alters the ability of agonist to bind to a receptor, or ability of receptor to undergo conformational change
why does the activation of a receptor does not directly induce a change in cell function
several proteins or small molecules are required to transduce this signal
what are the 3 common intracellular transduction pathways
- second messenger systems
- protein phosphorylation/ dephospho rylation
- GTP-binding proteins
what are second messengers
small molecules produced following receptor activation
what are some second messengers
cAMP, ca2+, IP3, DAG, cGMP
what is the purpose of second messengers
- provide signal amplification
- transmit signal from plasma membrane to cytosol
- activate downstream targets to further propagate the signal
what are the 7 cellular responses following receptor activation
- proliferation
- apoptosis
- contraction
- secretion
- movement
- differentiation
- altered metabolism
what are the main mechanisms that promote these types of changes
- modification of existing proteins
- changes in gene expression
how do we modify an existing protein
- phosphorylation/ dephosphorylation
- binding to second messenegrs
- binding to nucleotides
- binding to upstream signaling proteins which can alter the conformation of the target protein
what is protein phosphorylation
the addition of a phosphate group to a protein. performed by protein kinases
what is protein dephosphorylation
the removal of a phosphate group. performed by protein phosphotases
what does phosphorylation modify
- the function of the target protein
- enzyme activity
- binding site for additional protein
- protein dimerization
where does phosphprylation typically occur
on tyrosine, serine, and theronine amino acids
what does phosphorylation provide
molecular memory of pathway activation
what can protein kinases do to a receptor
be cytosolic, intrinsic, or attached
what do protein kinases do
add phosphates to specific amino acids, phosphorylate more than one target protein
how are protein kinases directed to a phosphorylation site
surrounding amino acid sequence
why do proteins have multiple phosphorylation sites
to allow for complex regulation of function
how can protein kinases be directly activated
following ligand binding to a receptor
what do protein phosphatases do
exhibit specificity where they remove phosphate groups from specific amino acids, dephosphorylate specific substrates
what is cAMP
cyclic adenosine 3’-5’-monophosphate
a cyclic nucleotide derived from ATP via adenylyl cyclase
what is adenylyl cyclase
a membrane bound protein with a cytosolic catalytic domain
what regulates cAMP’s activity
G-proteins and phosphorylation
what does cAMP regulate
the functions of cAMP-dependent proteins
- ex, protein kinase A, Ion channels
what is cAMP degraded by
nucleotide phosphodiesterases
what does plasma membrane contain
PIP2 (phosphatidylinositol-4,5-biphosphate)
PIp2 is broken down by phospholipase C to generate:
- inositol 1,4,5-triphosphate (IP3)
- diacylglycerog (DAG)
what is IP3
souble second messenger that can diffuse through cytosol
what is DAG
membrane-bound second messenger which activates protein kinase C
what are IP3 receptors
Ca2+ channels that open when bound to IP3, thereby releasing Ca2+ into the cytosol
what does IPs produced by phospholipase C bind to
IP3 receptors present on the ER membrane
what does IP3 stimulate
Ca2+ released from the endoplasmic reticulum
how does Ca2+ enter the cell from outside
voltage gated Ca2+ channels, Ligand-gated ion channels
how do voltage-gated Ca2+ channels let Ca2+ enter
open in response to membrane depolarization
how do ligand-gated ion channels let Ca2+ enter
open in response to ligand binding to a channel
how are cyrosolic cellular Ca2+ levels kept at rest
extremely low
what are trnasient elevations in ca2+ used as
intracellular signals
what is an example of a Ca2+ binding protein
calmodulin
Ca2+ that enters through ion channels or is released from the ER is removed from the cytosol through what
- Na+/Ca2+ exchanger and plasma membrane Ca2+-ATPase
- Sarcoplasmic/ endoplasmic reticulum Ca2+-ATPase
what do GTP binding proteins posess a binding site for
GTP and GDP
what happens when a protein is bound to GTP
protein is able to bind to downstream targets and regulate their functions
when is protein function inhibited
when GTP is dephosphorylated to GDP, protein function is inhibited
what are the two main forms of GTP-binding proteins
heterotrimeric G-proteins, small G-proteins
what is optogenetic Rac
a small GTPase that controls cell motility
what does GPCRs represent
the largest and most diverse class of plasma membrane receptors (approximately 1000 differnt ones in humans)
what do GPRCs do
regulate almost every physiological function in the human body
what are the differnt physical and chemical signals that GPCRs can detect
- photons of light
- ions
- lipids
- proteins/ peptides
- small organic molecules such as odorant’s, tastants, neurotransmitters
what do heterotrimeric G-proteins consist of
an α-subunit, a β-subunit and a γ-subunit
What are some of the key features of the α-subunits of heterotrimeric G-proteins
- a binding site for the intracellular region of the GPCR
- a binding site for GDP and GTP
- a binding site for the β subunit
- an intrinsic GTPase
what do we know about the β and γ subunits
- they remain as a dimer even during receptor activation
- they are largely interchangable for the different alpha subunits
- they are able to activate their own effectors
Where must effectors be located in order to interact with activated G-proteins
They must be membrane-bound because the G-proteins are embedded in the membrane
What factors affect the duration of GPCR signaling
- hydrolysis of GTP to GDP
- breakdown of second messengers
- phosphorylation/ dephosphorylation of proteins
- removal of Ca2+ from cytosol
what are the 3 main classes of G-protein α-subunits
-Gαs
-Gαi
-Gαq
What effector protein does each α-subunit regulate
Gαs – activates adenylyl cyclase (increasescAMP)
Gαi – inhibits adenylyl cyclase (decreases cAMP)
- Gαq – activates phospholipase C (produces DAG and IP3)
What transcription factor is commonly used to regulate gene expression via GPCRs
CREB (C)
How is this transcription factor (CREB) activated
phosphorylation by protein kinase A
what activated protein kinase A
cAMP
what is TrkA Receptor
plasma membrane receptor tyrosine kinase that is expressed by developing and mature neurons
what type of neurons are trkA receptors highly expressed in
sympathetic neurons
what is the TrkA receptor primarily activated by
nerve growth facor (NGF)
what is NGF secreted by
targets of sympathetic neurons
what happens when TrkA receptor produces more neurons than are required
- excess neurons are pruned
- NGF activation of TrkA leads to neuronal survival
- NGF also promotes axon development
Where are receptor tyrosine kinases found
Receptor tyrosine kinases are transmembrane proteins that are embedded in the plasma membrane
What conformation do they possess in the absence of a ligand
In the absence of a ligand, receptor tyrosine kinases exist as monomers
Briefly describe the mechanism responsible for receptor tyrosine kinase activation
- Ligand binds to receptor monomer which stimulates dimerization
- Each receptor has an intrinsic tyrosine kinase that is inactive when the receptor is a monomer
- Dimerization brings tyrosine kinases from the interacting receptors in close proximity
- Tyrosine kinases phosphorylate each other
- Additional tyrosines on the receptor are also phosphorylate
What are some of the consequences of receptor tyrosine kinase phosphorylation
- Increased activity of tyrosine kinase
- Generation of binding sites for additional protein
What are some of the downstream effectors of receptor tyrosine kinases
- Ras
- MAP kinase
What are some of the general characteristics of Ras
- Ras is a small G-protein
- Has GDP/GTP binding site and intrinsic GTPase
- Does not directly interact with receptors
What are some of the general characteristics of MAP kinase
- MAP kinases are protein kinases
- MAP kinase activity is increased by phosphorylation
- Exists as a monomer in the cytosol, where it can phosphorylate cytosolic transcription factors
- Following MAP kinase phosphorylation, it can become a dimer that is able to enter the nucleus to phosphorylate nuclear transcription factors
Where must a transcription factor be located to regulate gene expression
the nucleus
How could nuclear localization of a transcription factor be used to regulate gene expression
Nuclear localization could modify the ability of a transcription factor to enter the nucleus
what do Toll-like receptors do
- detect soncerved molecular sequences on foreign micro-organisms
- serve as one of the first methods of detecting invaders
- upon activation, receptors initiate intracellular signaling pathways that activate NF-κB
what is a TLR-4 ligand
a lipipolysaccharide component of the bacterial cell wall
what happens when something binds to TLR-4
receptor dimerization
(dimerization results in recruitment of accessory cytosolic proteins)
what happens during protein cleavage
- Receptor activation leads to cleavage of receptor or some type of inhibitor
- Releases/produces transcription factor which can then enter nucleus
what is the notch/delta signalling pathway
Notch is a plasma membrane receptor that is activated by a plasma membrane-associated protein known as delta
what happens when the delta binds to the notch receptor
the notch receptor undergoes two consecutive cleavages:
- cleavage of the extracellular region of the receptor
- cleavage of the intracellular region of the receptor
what is notch cleavage mediated by
an intramembrane protease known as gamma-secretase
The cleavage product derived from the intracellular region of the notch receptor acts as a what
transcription factor
what happens upon cleavage in the notch pathway
the intracellular domain of Notch translocates to the nucleus where it then alters gene expression
what are ligand-gated ion channels expressed by
skeletal muscle cells, certain neuronal populations and various other tissues throughout the body
why is the nicotinic acetylcholine receptor called “nicotinic”
because it can be activated by nicotine, in addition to acetylcholine
what does acetylcholine bind to
the extracellulr surface of the α subunits
Upon binding of agonist, the receptor undergoes a conformational change that does what
opens the central pore
the nicotinic acetylcholine receptor channel opening allows what cations to pass through
Na+, Ca2+ and K+
(Na+, Ca2+ enter the cell and K+ leaves the cell)
what is the new effect of nicotinic acetylchline receptor opening
cell depolarization
what are skeletal musce cells innervated by
lower motor neuron which release acetylcholine
what does activation of nicotonic acetylcholine receptor via acetylcholine cause
an opening of receptor pore where Na+ and Ca2+ enter the cell, causing a depolarization
what happens if depolarization reaches threshold in signaling pathways in skeletal muscle cells
voltage-gated Na+ channels are activated and an action potential is fired
action potentials activate a special type of voltage-sensor known as what
the dihydropyridine receptor
what does dihydropyridine receptor activation cause
Ca2+ release from te sarcoplasmic reticulum
what is the contractile machinery in skeletal muscle cells is made up of
actin and myosin proteins organized into sarcomeres
Interaction between actin and myosin is dependent upon what
cytosolic Ca2+ levels
what are the two regulatory proteins that actin is associated with
troponin and tropomyosin
what happens when Ca2+ levels are elevated
Ca2+ binds to troponin causing a conformational change that shifts tropomyosin away from the myosin binding site
what happens when myosin binds to actin
actin filaments are pulled towards the centre of the sarcomere, shortening cell length and generating force
what do F-actin bundles do
direct the initiation and orientation of lamellipodia through adhesion-based signaling
where are endocytic vesicles transported durind cell movement
lysosomes
where are exocytotic vesicles trnasported during cell movement
plasma membrane
where do immune cells migrate
to regions containing micro-organisms
where do fibroblasts migrate
injured tissues
What are some of the key characteristics of myosin?
- Myosin is a motor protein associated with the microfilament system of the cytoskeleton
- Myosin can bind to actin and uses energy from ATP hydrolysis to move actin filaments
- Myosin is a multimeric protein consisting of heavy and light chains
Each myosin heavy chain contains…
- A head region that is able to bind to actin and ATP/ADP and possesses an intrinsic ATPase
- A tail region that determines what cargo can be carried and helps form myosin dimers
- A neck region bound to myosin light chain
what is myosin V used to transport
- secretory vesicles
- organelles
- endocytic vesicles
Where does myosin V bind to, and how does it transport cargo
binds to its cargo via tail domain and transports cargo by crawling along actin filaments
what is Myosin II abundant in and what does it do
specialized contractile cells, such as skeletal muscle cells (also important in non-muscle cells). Associates with each other to form thick bundles with multiple myosin heads at each end
How does myosin cause cell contraction? What structures are involved and how are they altered during contraction?
In skeletal muscle, these thick myosin bundles (thick filaments) are strategically organized with respect to actin filaments (thin filaments) to generate structures known as sarcomeres
what is cell movement mediated by
microfilament reorganization and membrane recycling
what is the process of cell movement mediation
- Membrane protrusion
- Attachment of protruding membrane to extracellular matrix
- Movement of cytosol and membrane to leading edge of cell
what is membrane protrusion casued by
actin polymerization
what is nucleation promoted by
Arp2/3
where does elongation occur and what is it promoted by
occurs at positive end of F-actin and promoted by cofilin and profiling
how does actin synthesis work
- actin synthesis pushes membrane outwards, forming protrusions known as lamellopodia
How do the positive and negative ends of actin differ structurally
- Orientation of G-actin molecules is different
- Positive end has ATP-bound G-actin while negative end has ADP-bound G-actin
How are microfilaments synthesized in vitro
Nucleation, elongation and steady state
What conditions are required for spontaneous microfilament assembly
- The concentration of ATP-G-actin in solution must be high enough
- Critical concentration
What is the critical concentration?
The concentration of ATP-G-actin in solution that results in an equal rate of F-actin assembly and disassembly
Concentrations of ATP-G-actin above the critical concentration result in what
elongation of F-actin
Concentrations of ATP-G-actin below the critical concentration result in what
shortening of F-actin
How does the critical concentration differ between the positive and negative ends of F-actin
The Critical concentration at the positive end is lower than at the negative end
Which step in F-actin synthesis determines when and where this protein assembly will be produced
nucleation
What protein enables linear actin filaments to be synthesized
Formin
Define the term treadmilling
Refers to the cycling of G-actin molecules from the negative end of F-actin to the positive end
when does treadmilling occur
Occurs when the concentration of ATP-G-actin in solution falls between the critical concentrations of the positive and negative ends
What proteins affect treadmilling in vivo
Profilin, cofilin, and Capping proteins
What protein enables branched actin networks to be synthesized
Arp2/3
what are microtubules made up of
polymers of α-tubulin and β-tubulin
Describe the various structural elements of a microtubule
- αβ-tubulin forms linear polymers known as protofilaments
- Protofilaments associate laterally to form cylindrical, hollow tubes known as microtubules
what are protofilaments
α/β-tubulin dimers that associate with each other
what is the microtubule structure
13 protofilaments that associate with one another along the longitudinal axis of the tubule
what does the alignment of protofilaments provide
polarity to the microtubule. The positive end contains β-subunits and the negative end contains α-subunits
Describe the process of dynamic instability
–Microtubules undergo periods of growth, disassembly and re-growth
(Catastrophe and rescue)
What mechanism is responsible for dynamic instability in microtubules
The positive end of the microtubule terminates in a GTP-α/GTP-βtubulin cap
what must αβ-tubulin dimers contain to be added to the microtubule
GTP bound to both subunits
Why is dynamic instability useful to the cell?
- Microtubules will extend outward from MTOC, growing and retracting until appropriate target is reached
- Association between microtubule and target stabilizes microtubule
Where does microtubule nucleation occur
Microtubule organizing centres (MTOCs), Centrosome, and Basal bodies
what do animal cells and cilia/ flagella use as their microtubule-organizing centres
animal cells: centrosomes
cilia/ flagella: basal bodies
Nucleation in the centrosome is primarily achieved by what
γ-tubulin ring complex
what does the γ-tubulin ring complex do
- Serves as a template for microtubule synthesis
- Microtubule elongates at positive end
what do microtubule associated proteins do
Interact with microtubules to regulate their stability and growth
how can α- and β-tubulin molecules be post-translationally modified
- Glutamic acid residues, glycine residues or acetyl groups can be added
- Tyrosine can be removed
what are the purpose of microtubules
serve as tracks for motor proteins that transport various types of cellular cargo
what are the wo main microtubule-based motor proteins
kinesins: move cargo toward the positive end directly
dyneins: move cargo towards the negative end when intermediate protein is present
What are the main similarities between kinesins and dyneins
- Both transport cargo in the cell using microtubules as tracks
- Both use ATP to power their movement
what is the movement of kinesin and dynein
walks along a single protofilament within the microtubule using ATP hydrolysis to fuel this process
