Bsci330 Final Exam Spr 2010

Front 1

what are the parts of the endomembrane system?

Back 1

SER, RER, Golgi

Front 2

name the functionally divided pathways of the endomembrane system

Back 2

biosynthetic pathway

Front 3

what are the 5 parts of the biosynthetic pathway?

Back 3

1. proteins synthesized on RER
2. proteins modified in Golgi Complex
3. proteins sorted and delivered to various destinations (cytosol, organelles, etc)
4. proteins may be packaged into lumen of vesicles for secretion into EC space (secretory pathway)
5. proteins may be secreted continuously (constitutively) or be retained in secretory vesicles (granules) which fuse to PM and release contents in response to a stimulus

Front 4

what is the endocytotic pathway?

Back 4

moves proteins/materials from EC space and PM into (1) endosome vesicles, and then (2) lysosomes (where the contents are degraded)

Front 5

name 5 techniques of vesicle trafficking

Back 5

autoradiography, GFP labeling, biochemical analysis of fractions, cell-free systems, genetic mutants and knockouts

Front 6

describe autoradiography

Back 6

AR is used to detect and trace small molecules/proteins inside cells.
(1) tag the small molecule with a tracer atom.
(2) allow cells to take up molecule, mixed with untagged molecules
(3) fix the cells, make sections
(4) coat sections with a layer of photographic film emulsion
(5) radiation emitted by tagged mols. causes deposits of silver grains in areas where the molecule is concentrated
(6) view cells with silver grains with microscope. silver grains will show up as black in light or TEM.

Front 7

who was responsible for using autoradiography to investigate protein synthesis pathway?

Back 7

jamieson and palade - they studied the prot. syn. pathway in pancreatic acinar cells. they used PULSE CHASE labeling by first washing in radioactive AA, then in unlabelled AA.
they fixed cells at various times after the pulse labeling.

they found silver grains first localized to ER, then later, moved to golgi complex, then secretory vesicles.

Front 8

how is GFP regulated in terms of timing?

Back 8

use a promoter in the chimeric vector that is temperature sensitive, so only after a shock does the cell activate transcription of the foreign cDNA.

Front 9

how do you use biochemical analysis to investigate the protein synthesis pathway?

Back 9

homogenize (break them up) the cells with mechanical force, put them through a centrifuge as microsomes of different compartments have different densities, then put them in a density gradient solution to fraction them out. the molecular composition of the fractions can then be determined. an application is to find what other proteins bind to labeled newly synthesized proteins in each ER fraction as protein synthesis proceeds.

Front 10

compare RER's and SER's structure

Back 10

SER is typically a (1) tubular system where the space inside appears continuous and (2) lacks ribosomes. RER is an extensive organelle (1) composed mostly of cisternae (2) but the space appears continuous (3) ribosomes are attached on the cytosolic surface (4) RER is continuous with nuclear envelope outer membrane.

Front 11

what are the functions of the SER?

Back 11

functions vary cell-to-cell, BUT:
(a) synthesis of steroid hormones in gonad & adrenal cortex endocrine cells,
(b) detox in liver of many organic compounds
(c) large glycogen reserves are stored on outside of SER membranes
(d) storage of Ca2+ ions in SER lumen; their release triggers specific cell activities (SER contains high conc. Ca2+ binding proteins; regulated release of CA2+ from SER triggers responses like skeletal muscle contraction and fusion of secretory vesicles with PM)

Front 12

what are the functions of the RER?

Back 12

(a) synthesis and initial glycosylation (if applicable) of proteins:
(1a) proteins bound for excretion into the EC space
(1b) most integral membrane proteins

Front 13

what proteins are synthesized on the RER?

Back 13

proteins secreted from cells, integral membrane proteins, and soluble proteins that will reside within the compartments of the endomembrane system (ER, golgi complex, vesicles, lysosomes, etc)

Front 14

what proteins are synthesized on free ribosomes?

Back 14

proteins destined to remain in cytosol (enzymes of glycolysis, cytoskeletal proteins, etc), peripheral proteins of inner cell membrane surface (SPECTRINs, ANKYRINS), proteins that are transported into the nucleus, proteins to be incorporated into peroxisomes chloroplasts and mitochondria.

Front 15

describe the process of the creation of a protein by the RER.

Back 15

mRNA attaches to free ribosome; FR begins translation of mRNA; SRP (signal recognition particle) binds to signal on N-terminus and prevents further translation; SRP binds to SRP receptor protein on ER; SRP receptor releases SRP and then ribosome is attached to translocon (which allows for passage of polypeptide); polypeptide pushes aside a plug that covers the pore and translocates to completion; ribosome detaches; signal sequence is chopped by signal peptidase; molecular chaperones help re-form shape; PDI enzyme assists in disulfide bond formation from -SH groups.

Front 16

what happens to misfolded proteins in the ER?

Back 16

they are either covered by chaperone proteins, or they are sent through the process of reverse translocation, where they are digested by proteasomes, which are protein-degrading organelles.

Front 17

what are the steps for the creation of a glycoprotein?

Back 17

the glycoprotein goes through the protein creation steps of other proteins that exit the cell, but the ER lumen has enzymes which add carbohydrates to the protein, PDI which catalyzes the formation of disulfide bridges, and the proteins must first go through the ER-Golgi Intermediate Compartment (ERGIC) and thence the Golgi Complex.

Front 18

what is ERGIC?

Back 18

endoplasmic reticulum golgi intermediate compartment

Front 19

what is a VTC

Back 19

vesicle-tubular carrier - transport vesicles fuse in the ERGIC and move to the golgi complex via microtubule paths

Front 20

list functions of the golgi complex

Back 20

1. proteins are sequentially modified as they move from cis to trans face
2. protein's length may be trimmed by proteolytic enzymes
3. amino acids may be modified (hydroxylation of lysine and proline residues of collagen)
4.carbohydrate content of glycoproteins can be modified by stepwise enzymatic reactions
5. trans face of gogi system sorts and targets proteins for delivery by vesicles to their final destination (PM, lysosome, etc).

Front 21

what are the two models of the golgi complex's material movement and compare. what is the combined model?

Back 21

cisternal maturation model (CMM) - up to 1980s. cisternae form at the cis face by ER/ERGIC vesicle fusion and travel to the trans face, being altered along the way. It was believe that cisternae mature and change along the way; effectively "turning" into the next stack. transport vesicles carry resident golgi enzymes in a retograde direction (trans to cis).

vesicular transport model (mid 1980s-late 1990s)
cisternae remain in place as stable compartments held together by protein scaffold. cargo gets shuttled through the stack by vesicles budding from one cisterna and fusing vesicles to the next one.

now there is a combined model which believes in both forms of movement.

Front 22

what is movement in the golgi cis to trans direction called?

Back 22

anterograde movement; antonym is retrograde (trans to cis).

Front 23

how are vesicles distinguished in terms of their anterograde or retrograde movement?

Back 23

anterograde vesicles have COPII coating while retrograde vesicles have COPI coating. COPII and COPI are protein coats which cover the vesicle's bilayer.

Front 24

what is KDEL?

Back 24

its a sequence attached to ER resident proteins. it identifies proteins which must be shuttled back to the ER from the golgi complex via COPI-coated vesicles (retrograde movement).

Front 25

how do CDKs regulate the cell cycle?

Back 25

cdks wait to bind to cyclin (G1 or mitotic) to advance phases. cyclin levels are controlled by cycling degrading enzymes and by their localization (nucleus - active, cytosol - inactive). a cyclin causes a conformational change in the CDK that displaces a loop which once blocked the active site. the CDK is then free to phosphorylate serines and threonines. one type of cdk is cdc2 (=Cdk1) in yeast cells, which binds to the G1 cyclins to go from G1 to S, and the Mitotic cyclins to go from G2 to S. The cell cycle goes from M to G1 once the levels of cyclins drop.

Front 26

which cyclin has a NES and what does it do?

Back 26

Cyclin B1 has a nuclear export sequence (NES) that is responsible for it's export from the nucleus. once phosphorylated, the b1 cannot exit the nucleus (though it can still be imported), and thus an accumulation of cyclin b1 occurs inside the nucleus just before mitosis begins.

Front 27

how does phosphorylation of the cdk affect mitosis? name the steps.

Back 27

assume that cdc2 kinase is in its inactive form. Wee1 phosphorylates the Tyr15 residue which forces cdc2 to be inactive. CAK phosphorylates the Thr161 residue which must be phosphorylated for the cell to proceed in the cell cycle. An enzyme called cdc25 phosphatase then removes the phosphorous from the Tyr15 and the cdk becomes active. (figure has Thr161-cdc2 kinase-Tyr15). By the end of mitosis, the stimulatory Thr161 is dephosphorylated and the cdc2 kinase is back in it's inactive state.

Front 28

wee1 vs CAK vs cdc25 phosphatase. which is responsible for what in cell division?

Back 28

Wee1 phosphorylates Tyr15 (blocks mitosis).

CAK phosphorylates Thr161 (required for mitosis).

Cdc25 phosphatase dephosphorylates Tyr15 and allows the CDK to activate.

Front 29

which cyclin/cdk complexes are responsible for which part of the cell cycle?

Back 29

mid-g1 to mid-S:
first, D-Cdk4, D-Cdk6 (phosphorylates pRb which generates E, A cyclins)
next, E+Cdk2

S to S:
A+Cdk2 complex

S to G2 to M:
first, A-Cdk1
then, B-Cdk1

Front 30

what composes the checkpoint machinery?

Back 30

a sensor protein, a transmission protein, and an effector protein.

Front 31

what gene / kinase is responsible for stopping the cell cycle if DNA is damaged? what is the cascade pathway that is followed?

Back 31

the ATM gene (for a kinase) activates when damaged DNA is found. consequently, Chk2 is activated and phosphorylates p53, which promotes transcription and translation of p21. p21 then inhibits the kinase activity of a Cdk and thus entry into the S phase is denied. damaged DNA stops DNA replication.

Front 32

ATR vs ATM. go.

Back 32

ATR prevents the cell from going into M from G2 by activating Chk1 which phosphorylates cdc25 phosphatase and hence inactivates Cdk. ATM activvates Chk2 which phosphorylates (activates) p53, consequently p21, and p21 directly inhibits Cdk.

Front 33

name 4 types of stimuli that cells can respond to.

Back 33

1. mechanical (substrate adhesion, membrane distortion, sound).
2. light
3. heat
4. chemical (hormones, neutransmitters, olfactants, tastants, etc).

Front 34

how can cells respond to stimuli? name a few ways.

Back 34

1. activation or inhibition of certain intracellular enzymes
2. changes in cytoskeletal organization (activation of motile structures or adhesion to surfaces or cells)
3. changes in ion permeability through the PM (ie activating ion channels: changes membrane potential)
4. activation of gene transcription
5. activation of DNA synthesis or mitosis (pass through cell cycle transition points)

Front 35

describe the pathway of epinephrine (adrenalin).

Back 35

epinephrine binds to a receptor protein (beta-endrenergic) on the surface of the liver cells. the adrenergic receptor protein then activates a GTP-binding (g-protein) on the intracellular surface of the plasma membrane. the g-protein then activates ADENYLATE CYCLASE. adenylate cyclase catalyzes the production of cAMP from ATP. cAMP then binds to PKA (a protein kinase). PKA phosphorylates and inactivates glycogen synthase (halts glycogen synthesis from glucose) and instead phosphorylates and ACtivates phosphorylase kinase.
phosphorylase kinase phosphorylates and activates phosphorylase which stimulates the breakdown of glycogen into G1P which is then transformed into glucose.

Overall adrenalin increases glucose production and halts glycogen production. glucose can then be released to the bloodstream.

Front 36

what is beta adrenergic?

Back 36

receptor protein thats a member of the 7-alpha-helical transmembrane domain receptor family. members of the family have a similar tertiary core of 7 alpha-helical transmembrane segments with a central binding site for a small ligand molecule (extracellular side) and a binding site for the alpha helical subunit of a gtp-binding protein on the intracellular surface of the receptor protein.

Front 37

name some members of the 7-alpha-helical-transmembrane domain.

Back 37

beta adrenergic receptor (for epinephrine), hormone receptor proteins, neurotransmitter receptor proteins (metabotropic transmitter proteins that are not ion channels in themselves -- diff from ACh receptor in muscle cells), olfactory receptor proteins, taste cell receptor proteins...and more distantly...rhodopsin for light in the photoreceptor cells of eyes.

Front 38

describe g-protein movement in response to a signal.

Back 38

g-protein diffuses along PM until it hits an activated receptor protein like beta adrenergic, then the GDP on the g-protein is replaced with GTP. the alpha subunit of the GTP dissociates from the beta, gamma subunits and diffuses along the membrane until it hits a receptor protein such as adenylate cyclase.

Front 39

name ways in which the g-protein receptor protein complex is regulated....in terms of receptor desensitization

Back 39

1. dissociation of the ligand from the receptor
2. internal inactivation of the receptor protein-ligand complex (after a delay, the complex spontaneously becomes inactive)
3. phosphorylation of the complex by a kinase that leads to a binding of the protein "arrestin," which blocks activation of the g-protein.
4. receptor-mediated endocytosis.

Front 40

name ways in which the g-protein receptor protein complex is regulated....in terms of g protein inactivation.

Back 40

gtp bound to g-protein will become gdp eventually and alpha will rebind to gamma/beta subunits. gtp hydrolysis takes a few seconds and is regulated by GAP (GTP-ase accelerating proteins). GAP proteins thus control duration of response to ligand and total amount of second messenger product (ie: cAMP).

Front 41

describe the structure of PKA (protein kinase A) and how it works.

Back 41

PKA is composed of 2 regulatory and 2 catalytic subunits. the regulatory subunits inhibit the catalytic subunits until they are bound with cAMP. at this point, the catalytic subunits are free to react and activate phosphorylase kinase (which in turn turns on phosphorylase which turns glycogen into G1P). TWO cAMP molecules must bind to PKA simultaneously or else the catalytic subunits won't be activated -- it's a double check to avoid background cAMP activation.

Front 42

what is CREB and what's its function in protein synthesis?

Back 42

PKA phosphorylates the transcription factor CREB (cAMP-response element binding protein) which binds to a particular sequence in the promoter region of genes. CREB thus promotes transcription of these genes, which leads to new protein synthesis.

Front 43

in what ways can cAMP work?

Back 43

1. initiate enzyme activation (binding to PKA that leads to glycogen synthase inactivation and activation of phosphorylase kinase (which activates phosphorylase which stimulates breakdown of glycogen to G1P))
2. protein synthesis (IE: CREB)
3. changes in membrane potentials (IE: opening up ion channels which have a binding site for cAMP -- important in response of neurons to external stimuli)

Front 44

what does PKA bind to? what's the kinase cascade?

Back 44

phosphorylates (activates) phosphorylase kinase which then phosphorylates (activates) phosphorylase, the final effector enzyme catalyzing glycogen breakdown.

kinase cascades can also lead to enzyme or transcription factor activation.

Front 45

what's the purpose of kinase cascades?

Back 45

amplification -- each step leads to many new active kinase molecules catalyzed by a single prior kinase

(amplification also occurs by a single receptor activating many g-proteins)

Front 46

give two examples of when the phosphoinositide cascade is used.

Back 46

when ACh binds to smooth muscle and the smooth muscle contracts

and

when a foreign antigen binds to a mast cell in the blood and histamine is secreted.

Front 47

how does the phosphoinositade cascade work?

Back 47

response to a stimulus is mediated by the release of calcium ions stored in a cell and activation of PKC (protein kinase C). signaling pathway is based on metabolism of a minor class of membrane phospholipids - phosphoinositides.

1. ligand-receptor complex activates the g-protein (Gq) and the target enzyme phospholipase C-beta (PLC-Beta) is activated.

2. PLC-beta (at inner membrane surface) catalyzes splitting of PIP2 into 2 molecules; both of which are second messengers in cell signaling: IP3 (inositol 1,4,5-triphosphate) and DAG (diaglycerol).

There is an IP3 receptor on the smooth ER which in return for binding IP3, releases Ca2+ into the cytosol.

The Ca2+ ions bind to various target molecules and trigger specific responses and so act as intracellular messengers. However, since IP3 breaks down quickly into IP2, the effects are not as long lived as those of DAG/PKC.

Meanwhile, DAG becomes released in the plasma membrane and activates PKC, which phosphorylates many proteins and has a role in cell growth, differentiation, metabolism, transcription activation, and signal pathway regulation. overexpression of PKC can lead to cancer (mice).

Front 48

What are some of the roles of Ca2+ as an intracellular messenger?

Back 48

muscle contraction, cell division, exocytosis, endocytosis, fertilization, synaptic transmission, metabolism, cell movement.

Front 49

what are common types of Ca2+ emissions as a result of a stimulus like a hormone or neurotransmitter?

Back 49

1. sharp oscillations in Ca2+ ion concentration,
2. wave of Ca2+ release that spreads from one end of a cell to the other
3. localized and transient increase of Ca2+ ions in one part of the cell

Front 50

what is the relationship of [Ca2+] inside a cell versus outside?

Back 50

the innards of a cell have about 0.1uM while the extracellular space has about 1-2mM [Ca2+].

Front 51

what is the ryanodine receptor and where is it located?

Back 51

the ryanodine receptor is on the SER and is a calcium-activated calcium channel. when activated by cytosolic Ca2+, it releases even more Ca2+ into the cytoplasm, then shuts itself off.

Front 52

how do ryanodine receptors lead to Ca2+ spikes?

Back 52

ryanodine releases Ca2+ when it is met by Ca2+. a molecule such as IP3 can bind to its receptor on the SER and release little Ca2+ which binds to the ryanodine receptor. if the stimulus is kept then Ca2+ will be exposed to the ryanodine receptor over time regardless. The ryanodine receptor will release a lot of Ca2+ and close up, then again when activated, and again, etc. making waves.

Front 53

name 3 (or 4) ways in which calcium ions can directly activate or inhibit various enzymes.

Back 53

activation of troponin in skeletal muscle

enhancement of DAG's ability to activate PKC.

vesicle fusion and alteration of cytoskeletal structure and function.

open ion channels and cause membrane potential change.

can bind to calmodulin, which then binds to and activate enzymes and channels including Ca2+-calmodulin dependent kinase (very potent) which can initiate kinase cascades that activate transcription factors. Ca2+ can trigger protein synthesis this way. Calmodulin is highly conserved.

Front 54

what are the two types of photoreceptors in your eye and what are their functions?

Back 54

rods - dim light,
cones - less sensitive to light but the three types mediate color vision

Front 55

what's the structure of receptors in your eyes?

Back 55

behind the retina we have axons, ganglion cells, bipolar cells, which ultimately connect to either rods or cones.

rods and cones have a synapse, the nucleus, mitochondria, cilium, plasma membrane, cytoplasm, disk.

Front 56

how do rods and cones work?

Back 56

the outer segment of rods and cones have stacks of disks with high densities of visual pigment molecules which can provide a high probability of absorption of an incident photon.

the visual pigment molecules are composed of a chromophore (11-ci-retinal) covalently bonded to an opsin (receptor of 7-transmembrane family).

Front 57

what are the opsins in humans?

Back 57

we have 4.

1 - rods absorb green light
3 - cones absorb RGB light

Front 58

describe the transduction process in a rod.

Back 58

at rest (in darkness) cGMP levels are high and cGMP-gated Na/Ca channels in the PM are open. Na+ enters the cell and the cell is depolarized (ca. -20mV).

Then, light -> abs by rhodopsin -> g-protein (transducin) -> activation of phosphodiesterase (PDE) -> catalyzes hydrolysis of cGMP to GMP -> Na/Ca channels close -> rod hyperpolarizes to -70mV.

the neurons which make synaptic contact with the photoreceptor respond to a DECREASE in neurotransmitter emission!

Front 59

describe the pathway followed which results in a penile erection.

Back 59

1. "certain stimuli" cause ACh to be released from neurons which are near the blood vessels which control penile erection

2. Ach causes a rise in cytosolic Ca2+ ion concentration in endothelial cells via the phosphoinositide pathway and the activation of plasma membrane calcium channels

2B. Phosphoinositide pathway: ligand binds to receptor protein which activates G-protein (Gq) and phospholipase C-beta (PLC-beta) which catalyzes splitting PIP2 into IP3 and DAG. IP3 binds to a receptor on SER which releases Ca2+ into cytosol while DAG activates PKC.

3. Ca2+ ions activate nitric oxide synthase which converts arginine into NO.

4. NO diffuses out of endothelial cell and into neighboring smooth muscle cells.

5. NO activates guanylyl cyclase and cGMP is made from GTP.

6. cGMP activates PKG and effects other enzymes which lead to muscle relaxation, vasodilation, increased blood flow, and erection.

side note: Viagra works by inhibiting an isoform of PDE present in the penis (PDE destroys cGMP) and so works by increasing the lifetime of cGMP and thus promoting vasodilation and erection.

Front 60

instead of depending on a g-protein for signal transmission, insulin uses ___ proteins.

Back 60

RTK - receptor tyrosine kinase proteins.

Front 61

what is the structure of RTK proteins?

Back 61

4 subunits - 2 alpha, 2 beta.

alpha subunit has insulin binding site, beta subunit has a single transmembrane domain.

the intracellular domain of the beta subunit has an active catalytic site that phosphorylates tyrosine residues on other proteins as well as on itself.

Front 62

describe the insulin recognition pathway.

Back 62

insulin binds to the RTK's alpha subunits, which cause conformational changes to the beta subunits, who begin to phosphorylate tyrosine residues (including on themselves). They phosphorylate Insulin Receptor Substrates (IRS's) at tyrosine residues contained within specific sequences (PHOSPHOTYRONE MOTIF).

Then, proteins which have SH2 domains can bind to phosphorylated tyrosine sequences (eg on the RTK itself and IRS's). This SH2 binding alters the function of the SH2-laden proteins, which may become active enzymes and thus the RTK, IRS, w/their docked SH2 domain proteins make an assembly that can alter cell function.

Front 63

what are the types of enzymes with SH2 domains that bind to the phosphorylated tyrosine residues?

Back 63

PLC-gamma, which produces IP3 and DAG (note similarity to PLC-beta??)

Sos/Ras

PI-3-Kinase which phosphorylates the phosphoinositide lipid PIP2 into PI(3,4,5)P3. PI(3,4,5)P3 activates protein kinase B (PKB) which phosphorylates proteins that promote glucose uptake and glycogen synthesis.

Front 64

what is another example of an RTK besides the one for the insulin receptor? describe structure.

Back 64

it is a monomer, and when bound with a ligand, joins another monomer and they form a dimer. as they are active, they autophosphorylate and then can bind to SH2-domain proteins.

Front 65

describe the Ras pathway.

Back 65

Ras is a small (monomeric) GTP-binding protein, bound by a lipid anchor to plasma membranes.

It is activated by phosphorylated (activated) RTK's via:

1. phosphorylated RTK binds SH2-domain adaptor protein Grb2/Sos, recruiting Sos to the inner leafleft of PM, where it meets Ras.

2. Grb2/Sos binds to Ras and Sos and promotes exchange of GTP for GDP on Ras (thus Ras activates ie: "Ras-GTP")

3. Ras-GTP binds a kinase called Raf. Raf then begins the MAP kinase cascade.

4. The MAP kinase cascade results in the activation of MAPK, which enters the nucleus and phosphorylates (activates) transcription factors like Elk-1.

5. The activated TFs promote synthesis of Fos and Jun.

6. Fos and Jun form another TF, AP-1, which promotes synthesis of cyclins and genes involved in cell growth and division. MAP kinase cascade can also phosphorylate other TFs like CREB.