BIOM20001 - Topic 3 Master

cell topology (def)

space constructed by an enclosed single membrane with an internal space or lumen

cell membrane leaflet types (2)

cytosolic leaflet → side facing inside of cell

luminal extra-leaflet → side facing outside of the cell

methods of protein movement (4)

protein translocation

gated transport

vesicular transport

engulfment

methods of protein movement - protein translocation (4)

cytosol to plastids, mitochondria, peroxisomes, ER

methods of protein movement - gated transport (1)

between nucleus and cytosol

methods of protein movement - vesicular transport (6)

ER to peroxisomes

between ER and Golgi

between Golgi and late endosome, early endosome, secretory vesicles

early endosome to latte endosome

between early endosome and plasma membrane

secretory vesicles to plasma membrane

methods of protein movement - engulfment (2)

cytosol to nucleus

plasma membrane to endosome

proteins are sorted via….

sorting signals

read by sorting receptors which deliver to target location

protein sorting signal types (2)

signal sequences for translocation

signal patches for nuclear and vesicular transport

main function of ER (with secondary)

biosynthesis of lipids and proteins

also stores intracellular calcium ions which can be rapidly mobilised to regulate various cell signalling responses

signal peptidase function - ER translocation

closely associated with translocator and cleaves off signal sequence during translation

mature protein is released into the lumen of the Er immediately after synthesis is completed

single pass transmembrane proteins - N terminal domain retained on cytosolic side (2)

favoured for proteins with very long or folded internal domains

transmembrane segments with flanking amino acids that have a net positive charge on the N terminal side

single pass transmembrane proteins - C terminal domain retained on cytosolic side (1)

favoured for transmembrane segments with flanking amino acids that have a net positive charge on the C terminal side

what signal sequence do all proteins bound for the ER have

ER signal sequence → often at N terminus

ER resident proteins will have a ER retention sequence → KDEL or HDEL

benefit of having an ER signal sequence

ER resident proteins sent to the Golgi for further modifications or incorrectly sent out of ER can be redirected to Er via sequence

what sequence in transmembrane proteins is recognised

at least one hydrophobic transmembrane sequence is recognised by translocator protein → passed through lateral gate to be embedded in membrane

N-linked glycosylation (def)

complex sugar group is added to the side chain NH2 group of an asparagine residue

quality control mechanism

where is the energy required for mitochondrial translocation from (3)

ATP hydrolysis

energy from membrane potential

energy form redox potential

why is translocation not required for transport between nucleus and cytosol

lumen of nucleus is topologically equivalent to the cytosol

nuclear pores allow for communication between nucleus and cytosol

nuclear pore structure (4)

Scaffold and channel nucleoporins (transmembrane proteins) forms ring around central nuclear pore

Channel nucleoporins extend mesh of disordered domains into central pore

Cytosolic fibrils extend form the scaffold nucleoporins into the cytosol

Cytosolic fibrils also extend from the scaffold nucleoporins into the nucleus → form nuclear baskets

regulation of nuclear import and export is achieved by…

differential localisation of Ran GDP in the cytosl and Ran GTP int he ncuelus provides directionality to nuclear transport

Ran GDP is abundant in cytosol due to presence of Ran GAPs in cytosol

Ran GTP is abundant in nucleus due to presence of Ran GEFs

Rand GDP is transported down concentration gradient into the nucleus from the cytosol → converted to Ran GTP by Ran GEF etc

prospective nuclear and cytosolic protein signals

cytosolic will have cytosol localisation signal

nuclear will have nuclear localisation signal

signal is recognised by receptors

how is dissociation of cargo from transport receptors achieved

GTP to GDP exchange encourages cargo offloading in the cytosol

GDP to GTP exchange encourages cargo offloading int he nucleus

transport vesicles - coat protein types (4)

Clathrin mediates transport from the plasma membrane via endocytosis and transport form the Golgi and endosomes

COPI facilitates reverse Golgi to ER transport

COPII facilitates forward ER to Golgi transport

Retromer facilitates endosome to Golgi

how is vesicle structure determined - with example

shape depends on molecules that make up the coat

clathrin forms triskelion of three heavy chains and three light chains → multiple triskelia come together to form deformed soccer ball-like structure

how is a vesicle pinched off the donor membrane

membrane bending and fission proteins constrict the neck and eventually pinch off the vesicle

how is a vesicle pinched off the donor membrane - example protein

dynamin wraps around the neck and separates the vesicles

GTP dependent

how is fusion of vesicles with target membranes regulated (what molecules - 2)

Rab proteins → monomeric GTPases

SNARES → V and T

fusion of vesicles with target membrane - step summary (3)

1. specific Rab proteins localise to specific organelle membranes → presence of specific Rab proteins creates and can change the identity of an organelle

2. GTP-bound Rab will insert into a membrane and interact with complementary Rab-effector proteins

3. Rab effector pulls vesicle in to allow SNARES to interact → V on vesicle, T on target membrane

transport from ER to Golgi - protein coat type

COPII binds to adaptor proteins which recognise either the lipid surface of the membrane or the proteins that will be the cargo in the forming vesicles

GTP-dependent switches provide energy for initial recognition

transport from ER to Golgi - vesicle formation steps (5)

1. inactive soluble Sar-1-GDP binds to Sar-1-Gef in ER membrane → exchanges GDP for GTP

2. conformational change exposes amphiphilic helix which inserts into the cytoplasmic leaflet of the ER membrane → membrane bending initiated

3. Sec23 adaptor protein binds to active Sar-1, Sec24 binds to cargo receptor cytosolic tail

4. COPII coat proteins bind to adaptor proteins to form outer shell → budding initiated

5. membrane fission event pinches off vesicle

COPII vesicles merge to form…

vesicular tubular clusters → travel along microtubules towards the cis Golgi network

types of Golgi to endosome and cell membrane secretion (2)

constitutive → default pathway and operates in all cells

regulated → requires specific signalling and occurs in secretory cells

diverge at trans-Golgi

how is content in secretory vesicles concentrated

vesicle fusion and membrane recycling

immature vesicles fuse with other vesicles to form mature secretory vesicles → small parts of membrane pinched off and returned to Golgi

mature cells remain around plasma membrane until they receive a signal to release their contents

lysosome organelle type

main organelle for controlled intracellular degradation

lysosomal pathway

part of secretory pathway that delivers material from various molecules to be degraded in lysosomes

enzymes in lysosomes - origin of synthesis

acid hydrolases synthesised by rough ER bound ribosomes and processed into the rER and Golgi

enzymes in lysosomes - function

only function in acidic environments → ensures that they only function within the lysosome and prevents them from degrading other molecules if they escape

lysosomes - method of degradation

vesicle carrying cargo that needs to be degraded fuses with lysosome

material can be from within the cell (autophagy) or from outside the cell via endocytosis or phagocytosis

lysosomal protein tagging (4)

proteins tagged with N-linked oligosaccharide for protein folding

oligosaccharide phosphorylated in the cis Golgi network

mannose-6-phosphate tages potein for lysosome targetting

M6P receptor recruits adaptor proteins which bind M6P-modified lysosomeal hydrolases

dissociation of lysosomal hydrolases from M6P receptors and return of receptors to the Golgi

at lower pHs of endosome, hydrolases dissociated from receptors

empty receptors retrieved by retromer-coated vesicles and sent back to trans Golgi network

phosphate removed form M6P attached to hydrolase → prevents hdyrolases from being sent back to Golgi

exocytosis (def)

process of fusing transport vesicles with plasma membrane to release its content intot he extra cellular space

endocytosis (def)

process of plasma membrane internalising and forming a transport vesicle with the content derived from the extracellular space

endocytosis - pinocytosis

endocytosis of smaller molecules and fluids → clathrin coated vesicles ebola and salmonella use macropinocytosis → clathrin independent

endocytosis of LDL to form cholesterol (7)

1. LDL is detected by LDL receptors on the plasma membrane

2. Specific adaptor proteins are recruited by the receptors

3. Clathrin recruited to form clathrin-coated vesicle

4. Clathrin dissociates and naked vesicle fuses with an early endosome

5. LDL dissociates from its receptor and early endosome matures into late endosome

6. Late endosome fuses with a lysosome

7. Hydrolytic enzymes in the endolysosome degrades the LDL into free cholesterol -> can be used to build new membrane

   • Unbound LDL receptor is transported back to plasma membrane

multivesicular bodies (def)

vesicles within vesicles

multivesicular bodies - benefit (2)

1. frees up ubiquitin → binds to signalling receptors to prevent it from further signalling, intralumenal vesicle formation allows it to be released and recycled as receptor can no longer signal

2. allows for degradation of all parts of cargo → intralumenal vesicle membrane can be degraded allowing access to cytosolic regions of receptor

meaning of two cellular components being topologically equivalent

protein can cross form one compartment to the other without physically cross a membrane

during glycosylation of an extracellular cell surface receptor, where would the extracellular domain of the glycoprotein project into

lumen of er

principles of cell signalling (4)

1. binding of extracellular signalling molecule to the receptor protein will activate the protein

2. activated receptor will activate one or more intracellular signalling pathways involving a series of intracellular signalling proteins

3. one or more of the intracellular signalling proteins will alter the activity of effector proteins

4. altered activity of effector proteins will alter the activity of the cell by inducing a particular metabolic pathway

types of intercellular signalling - list (4)

contact dependent

paracrine

synaptic

endocrine

intercellular signalling - contact dependent

cells in direct membrane to membrane contact

membrane bound signal molecule on a signalling cell can bind to a receptor on the target cell

intercellular signalling - paracrine

local mediators released from signalling cell can bind to a receptor on the target cell

intercellular signalling - paracrine example

autocrine signalling

signalling and target cell are the same

intercellular signalling - synaptic

neurons transmit signals electrically along their axons and then release chemical neurotransmitters at the synapses to act on the target cell

intercellular signalling - endocrine

secrete hormones into the blood stream which act on target cells throughout the body

location of intercellular signalling receptors (2)

on the cell surface or intracellularly

location of receptor effects how the signal is transmitted into the cell

intercellular signalling receptors - hydrophilic signalling molecules

majority cannot pass hydrophobic cell membrane → bind to cell surface receptors which can direct signalling pathways within the cell

intercellular signalling receptors - small signalling molecules

can diffuse across the plasma membrane and into the cell to bind to receptors in the cytosol or int he nucleus

mostly hydrophobic so need to be transported via carrier protein in the bloodstream or within the extracellular environment and then dissociate before cross in the membrane

signalling by phosphorylation - summary

protein kinase covalently adds phosphate from ATP to the signalling protein

protein phosphatase removes the phosphate

phosphorylation and dephosphoryaltion can both activate or inactive a signalling pathway

signalling by GTP binding - summary

GTP binding protein is induced to exchange its bound GDP for GTP → always activates protein

GTP binding protein can inactivate itself by hydrolysing its bound GTP to GDP

structure of GPCRs

present in all eukaryotes with the similar structure of 7 transmembrane segments

what do GPCRs respond to (6)

hormones

neurotransmitters

local mediators

light photons

amino acids

fatty acids

true or false - the same signal can only activate one GPCR depending on cell type

false

same signal can activate multiple GPCRs depending on cell type

acetylcholine activates 5 GPCRs with different effects on different cells depending on which GPCR and ion-channel receptor it binds to

G protein subunits and location within the cell (3)

trimeric binding proteins with three subunits

alpha and gamma = membrane bound

beta = cytosolic

active and inactive state of G proteins difference

active: alpha is GTP bound

inactive: alpha is GDP bound

activation of G protein steps (5)

1. Signal molecule binds to GPCR induces conformational change of receptor

2. Activated GPCR can bind and alter the conformation of the trimeric G protein

3. AH domain of G protein opens up to expose nucleotide binding site -> promotes dissociation of GDP

4. GTP binding to AH domain promotes closure of nucleotide binding side -> triggers conformational changes that dissociates alpha subunit from receptor and activates gamma subunit

5. GTP bound alpha subunit and activated beta gamma subcomplex both regulate the activity of downstream signalling molecules

role of GPCR in amplifying intracellular respone

as signal molecule stays bound to GPCR, receptor can activate many G protein molecules

what is adenylyl cyclase targeted by and what is its role

common target for G proteins

catalyses synthesis of cyclic AMP from ATP

effect of cholera on G proteins

cholera toxin lead sot overactive G protein → increased activation of adenylyl cyclase and up-regulated chlorine channel opening which leads to diarrhoea

effect of serotonin on G proteins

serotonin acts through GPCR to cause rapid rise or fall in intracellular concentrations of cyclic AMP

depends on if G protein is negatively or positively coupled with adenylyl cyclase

effect of GPCR on gene expression via PKA - activatation of GPCR to activation of PKA (4)

1. activated GPCR activates G protein

2. activated alpha subunit activates adenylyl cyclase

3. adenylyl cyclase catalyses production of cAMP

4. cAMP binds to regualtory subunits of PKA tetramer to induce conformational change that causes dissociation of regulatory subunits and activates kinase activity of catalytic subunits

effect of GPCR on gene expression via PKA - PKA in nucleus to gene expression (2)

5. active type 1 and 2 PKA can enter nucleus via nuclear pore and phosphorylate an inactive CREB protein

6. activated CREB protein can bind associated CREB binding proteins and activate target gene expression via cyclic AMP response elements on DNA

effect of GPCR on gene expression via PKA - requirement

2 or more cyclic AMP molecules need to bind to regulatory subunits

sharpens response of kinase to changes in cAMP concentrations

types of PKA - location (2)

type 1: mainly in cytosol

type 2: bound via its regulatory subunits and anchoring proteins to the plasma, nuclear and mitochondrial outer membrane and microtubules

how are GPCRs related to vision and smell

both depend on GPCR regulated ion channels

photoreceptor structure

composed of rods and cones

rods detect dark and cones detect colour and bright light

outer segment contains photoreceptor discs which express rhodopsin GPCRs

Ion flux - reaction to light (5)

1. Light enters the outer segment of photoreceptor cells -> rhodopsin GCPRs in photoreceptor discs absorb light and become activated via conformation change to linked protein retinal

2. G alpha subunit of G protein transducin activates cyclic GMP phosphodiesterase

3. Activated cGMP phosphodiesterase hydrolyses cyclic GMP hydrolysis -> decrease in concentration of cGMP

4. Closure of cGMP gated sodium channels -> hyperpolarisation of cell and decreased rate of neurotransmitter release from synaptic region

5. neurotransmitter inhibits many of the postsynaptic retinal neurons → illumination frees neurons from inhibitions and thus excites them

ion flux - reaction to darkness (2)

1. Rhodopsin kinase phosphorylates and inhibits rhodopsin

2. arrestin binds phosphorylated rhosopsins and further inhibits its activity

3. regulators of G proteins (RGS) proteins bind transducin G protein and hydrolyse GTP to GDP to inactive it

photoreceptor cells - bipolar cells

activated bipolar cells transmit signals to retinal ganglion cells → signals to brain

photoreceptor cells on bipolar cells

can signal to ON or OFF bipolar cells

photoreceptor cells on bipolar cells - dark

photoreceptors release glutamate → inhibits ON bipolar cells and excites OFF bipolar cells

photoreceptor cells on bipolar cells - light

photoreceptor hyperpolarisation prevents inhibition of ON bipolar cells and inactivates OFF bipolar cells

structure of receptor tyrosine kinases

single transmembrane domain within plasma membrane

variable extracellular and intracellular domains → tyrosine kinase domain is always in cytosol

kinase domain can be interrupted by kinase insert region → essentially two tyrosine kinase domains

summary of RTK activation (5)

1. Signal protein binds to inactive receptor tyrosine kinases

2. Dimerization of RTKs brings kinases together

3. Trans-autophosphorylation -> each receptor phosphorylates and fully activates the other

4. Further phosphorylation generates binding sites for other intracellular signalling proteins

5. Signalling proteins bind to specific docking sites and are activated -> can relay signal to other downstream effectors

binding of signalling proteins to tyrosine kinase domains

majority do so via SH2 domains and bind to specific phosphotyrosines

usually have a SH3 domain which can bind to other intracellular signalling molecules

activated RTK to activated Ras protein - steps (4)

1. activation of RTK → adaptor proteins binds to specific phosphorytosine

2. adaptor proteins bind and activates GEF via SH3 domain

3. GEF encourages Ras-GDP to hydrolyse bound GDP and thus bind GTP → active

4. activated Ras-GTP can induce downstream signalling pathways

limitation of RTKs and Ras proteins and solution

only active for a short period of time

initial signalling event is converted into longer-term signalling event via activation of MAP kinase signalling cascade

MAP kianse signalling cascade - summary (4)

1. Active GTP bound Ras activates MAP kinase kinase kinase (Raf in mammalian cells)

2. Raf activates MAP kinase kinase (Mek)

3. Mek activates MAP kianse (Erk)

4. Active Erk phosphorylates other proteins and gene regulatory proteins

benefit of dimerisation of RTKs

allows inhibition of signalling using domain negative receptors

if dominant negative receptor is over expressed in a cell, will replace RTKs → when ligand binds to cause dimerization trans-autophosphorylation does not occur → no signal transduction

dominant negative receptor - def

functioning extracellular domain and transmembrane domain but truncated intracellular kinase domain → no tyrosine kinase domain so no catalytic activity

TGFß composition (2)

type 2 receptor homodimer = specific for each type of ligand and constitutively active

type 1 receptor homodimer = activated via phosphorylation by type 2 receptor

effect of activated TGFß receptor complex on gene transcription (2)

1. activated TGFß binds and phosphorylates SMADs

2. Trimeric SMAD complex forms and translocates to the nucleus to form transcription regulatory complex through interactions with transcriptional regulators

common target genes mediated by TGFß signalling (7)

Inhibition of proliferation

Cell specification and differentiation

Extracellular matrix production

Epithelial to mesenchymal transformations or fibrosis (can lead to cancer metastasis)

Cell death

Tissue repair

Immune cell regulation

activated TGFß receptor and clathrin mediated endocytosis - SMAD activation

most of SMAD activation occurs in early endosomes and requires SMAD anchor for receptor activation (SARA)

inactivation of activated receptor complex requires caveolae mediated endocytosis

receptor ubiquitylation and degradation in proteosomes occurs

Wnt signalling pathways (3)

PCP signalling

ß-catenin-dependent signalling

Wnt-Ca2+ signalling

what cells use Wnt signalling

most commonly active in epithelial cells

also occurs in neurons, lymphocytes, muscles and other cells

ß-catenin role - adherens junctions

component of epithelial adherens junctions

connects actin filament bundles and anchors cells to each other

adherens junction reassembly’s effect on ß-catenin

adherenes junctions = dynamic

reassembly of adherence junctions will result in ß-catenin release into the cytoplasm

ß-catenin - Wnt signal on destruction

under normal conditions, cells does not want ß-catenin in cytosol → destroy via proteolysis

if Wnt signal via ß-catenin dependent pathway → ß-catenin stabilised