Membrane Dynamics and Function Flashcards
what are the 4 general ways substances can be exchanged across the PM?
lipophilic substances dissolve in PM, channels allow materials to diffuse through, transporters couple transport to energy source to move materials against concentration gradient, in eukaryotes vesicles used to import and export materials
why are lipophilic chemical signals made on demand?
cells can’t retain them as they can dissolve in and pass through membranes
how are non-lipophilic messengers recognised by cells?
bind to either extracellular binding site on a channel or on an allosteric membrane-spanning protein
describe the nucleus
home to genome, separated from cytosol by double membrane penetrated by complex nuclear pores (perhaps 4000/nucleus) through which materials guided in and out
describe the endoplasmic reticulum
most abundant and invasive organelle, makes contact with most other organelles in cell, encloses ER lumen where proteins mature and Ca2+ stored
what does the RER have that the SER doesn’t?
studded with ribosomes (site of protein synthesis)
role of ER?
lipid synthesis, steroid synthesis, protein maturation, Ca2+ storage, RER= protein synthesis
describe the Golgi apparatus + its roles
stacks of tubules linked to ER by vesicular trafficking. important roles in maturation/glycosylation of proteins + their dispatch to other intracellular destinations
how many mitochondrial genes are there? what do they do?
- encode tiny fraction of mitochondrial proteins. are transcribed and translated within mitochondria using machinery reminiscent of bacteria.
how are proteins dispatched to different locations after forming on a cytosolic ribosome?
address labels within primary sequence dispatch proteins to different destinations
what causes fully folded proteins to move to the nucleoplasm through nuclear pores?
nuclear localisation signals
why do all proteins that don’t go to the nucleus have to remain partially unfolded before reaching their final destinations?
the rest all have to cross a membrane to reach their final destinations, need to be partially unfolded to be threaded through protein pore within target membrane
state of proteins destined for mitochondria and peroxisomes before being dispatched from ribosome?
fully translated
what is co-translational targeting, what destination usually has proteins that use co-translational targeting?
when the protein is dispatched before translation is complete. proteins destined for ER usually use this
what is the nuclear localisation signal?
stretch of around 6 positively charged residues anywhere in primary sequence of proteins, recognised by importin, directs reversible passage through nuclear pores
address label for proteins destined for peroxisomes?
C-terminal sequence SKL-e recognised by PTS1 receptor
what causes Zellweger syndrome?
non-functional PTS1 receptor for proteins targeted to peroxisomes
what are the 4 destinations in the mitochondria for proteins?
inner and outer membranes, inter-membrane space, matrix
what is the address label for proteins destined for the mitochondrial matrix?
N terminal amphipathic helix recognised by a chaperone protein
is targeting to peroxisomes and mitochondria reversible or irreversible?
irreversible
where can proteins sent to the ER be sent to from there?
Golgi, lysosomes, secretory pathway, plasma membrane
signal sequence for proteins sent to the ER lumen vs ER membrane?
hydrophobic signal sequence (at least 8 continuous hydrophobic residues) at the N terminal for ER lumen proteins, internally for integral membrane proteins. recognised by the SRP
what is the SRP?
signal recognition particle- a large protein-RNA complex which recognises the hydrophobic signal sequence of proteins sent to the ER
what happens when the SRP detects the hydrophobic signal sequence on an incompletely translated protein?
translation arrests, SRP-nascent peptide chain associates with SRP receptor on ER membrane. SRP receptor associates with translocon (protein channel in ER membrane), checks signal sequence of peptide. nascent peptide chain inserted in appropriate orientation into translocon (this is final orientation). SRP is released
what happens when the SRP releases the translocon?
translation resumes and the growing protein is threaded through the translocon which continues to survey the growing protein looking for stretches of hydrophobic residues to form membrane-spanning domains- these residues retained in translocon until all TMDs are fully assembled then pass into ER membrane
what happens as the growing peptide emerges through the translocon into the ER lumen?
it is scrutinised by enzymes, protease cleaves any N-terminal signal sequence, N-linked glycosylation occurs, chaperone proteins (e.g. BiP) pull the growing peptide into the ER lumen, help it fold, other enzymes make additional modifications
what enzyme catalyses N-linked glycosylation, where and to which residues?
oligosaccharyl transferase, in ER lumen, to Asn residues within the sequence Asn-X-Ser/Thr
what is ERAD?
ER-associated degradation
why is ERAD necessary?
some proteins are damaged in the ER lumen, must be removed or they could aggregate and clog the system
what does ERAD do?
allows misfolded proteins to be returned to cytosol via pore associated with a ubiquitin ligase that attaches Ub to Lys residues on the protein marking it for degradation
what is defective in Parkinson’ disease?
Parkin which encodes subunit of ubiquitin ligase, prevents ERAD
how many ER proteins are marked for degradation?
more than 30%- system plays safe
what happens in the most common cause of cystic fibrosis?
CFTR protein is degraded by ERAD before it can reach plasma membrane to mediate Cl- transport
how does CMV hijack the ERAD pathway?
expresses protein that associates with the major histocompatibility complex (MHC) that would normally display proteins on cell surface to indicate cell is infected so virus can hide within the cell
is targeting proteins to the ER from the cytosol reversible?
no
what happens to the folded glycosylated proteins produced in the ER?
collected into COPII vesicles, conveyed to the cis Golgi
what happens to glycosylated proteins within the cis-Golgi?
sugar structures are trimmed and modified, proteins destined for lysosomes have sugars modified to include mannose-6-phosphate.
what do vesicles travel via?
microtubules
what are Rab proteins?
small G-proteins which act as identity labels for organelle membranes
what happens in the Rab cycle?
Rab-GDP recruited to an intracellular membrane, activated if host has specific proteins needed to make it shed GDP and attach GTP. if another organelle recognises the Rab-GTP and has the proteins needed to hydrolyse the GTP, does this, releasing the Rab, interacting with the original membrane
what do COPII coat proteins do?
bind to specific cytosolic sequence of proteins embedded in ER membrane, collect them into clusters which are pinched off as small COPII vesicles,
that move along microtubules to the cis-Golgi and shed coat on the way to unmask underlying identity labels
what proteins mediate fusion of COPII vesicles with the cis-Golgi?
SNARE (SNAP receptor) proteins
how do SNARE proteins work?
draw the 2 organelle membranes close together to mediate their fusion
what carries cargo back from the Golgi to the ER?
COPI vesicles
what recognises luminal cargo?
KDEL sequence that binds to membrane protein KEL receptor which assembles a COPI coat. vesicles pinched off, recognition and fusion at ER lumen
function of the trans Golgi?
sorts proteins to different destinations
how are soluble proteins destined for lysosomes recognised by the trans-Golgi?
by their M-6-P modification acquired in the Golgi
what are M6P receptors?
membrane proteins that recognise M6P at their luminal surface, have short sequences that assemble a clathrin coat at their cytosolic surface
what recognise the proteins bound to M6P receptors?
late endosomes which cause cargo to dissociate from the M6P receptors, pass them on to lysosomes
what happens to the M6P receptors after endosomes remove their cargo?
they are sorted into vesicles and returned to trans Golgi
what underlie an array of lysosomal storage diseases?
genetic deficiencies in the enzymes that are uniquely expressed in lysosomes
treatment option for Gaucher disease (a lysosomal storage disease)?
administering M6P modified form of enzyme that is missing in the disease, which can be endocytosed by the M6P receptor and delivered to lysosomes
purpose of endocytosis?
allows import of materials from outside cell and of endogenous materials from PM
how is cholesterol imported from the plasma?
all cells express low-density lipoprotein receptors, bind to ApoB-100 component of LDL. cytosolic domain of LDL receptor has sequence that interact with AP2 and thereby clathrin, assembles LDL receptors with their LDL cargo into clathrin coated pits for trafficking to endosomes- in acidic lumen of endosome LDL dissociates from receptor + is recycled to PM, passes to lysosomes where ApoB-100 is degraded, freeing cholesterol to pass out of lysosome
what is the fastest method of communication across the plasma membrane?
ion channels
why is Ca2+ the ion used to regulate cellular activity?
the channels affect membrane potential, MP changes must be transduced into a messenger that enters the cell- this messenger is Ca2+ which can regulate many things such as release of neurotransmitter
how do nicotinic ACh receptors in muscle work?
when 2 ACh molecules bind to the 2 alpha subunits, conformational change opens ion channel in protein which is equally permeable to Na and K
structure of nAChRs?
pentameric with 2 alpha subunits, 1 beta, 1 delta, 1 gamma
abilities required of ion channels?
must be able to open, close and select between ions
common plan of voltage gated cation channels?
24 membrane spanning (4x6) regions arranged around central pore
which voltage gated cation channels are formed from 1 protein chain, which is formed from 4 protein subunits?
Na+ and Ca2+ both formed from 1 chain, K+ formed from 4 proteins with 6 domains each
what provides selectivity in voltage gated cation channels?
narrow ‘selectivity filter’ through which cations must pass in single file. allows surrounding amino acid residues to interact with permeating cation
structure of selectivity filter of Ca2+ channels?
2 Ca2+ binding sites, each can bind Ca2+ selectively and tightly, when both occupies electrostatic repulsion between the 2 divalent cations causes 1 to be dislodged
what closes ion channels?
channel residues at cytosolic tip of pore-linking helix (TMD6) come together like top of a teepee, occlude the channel
what is the voltage sensor in VG ion channels? how does this lead to channels opening?
4 positively charged residues in TMD4 of each of 4 channel domains moves outwards in plane of membrane when PM depolarises, pulls on short cytosolic helix (links TMD4-5) causes twisting of pore-lining helices and splaying of teepee so ions can pass
general features of signalling pathways initiated by allosteric receptor proteins?
receptor protein is what transmits information through its structure across PM, information passes onward through sequence of protein-protein and protein-small molecule interactions. many steps in pathway = amplification, allows cells to be very sensitive to extracellular stimuli. all steps in sequence are reversible. scaffold proteins arrange proteins into spatially-organised macromolecular complexes that allow effecive + specific information transfer. signalling pathways integrate information from other pathways
how do receptor tyrosine kinases work?
extracellular signal binds, causes receptor to dimerise, dimerisation activates cytosolic tyrosine kinase activity on receptor, allows each subunit to transphosphorylate Tyr residues on the other. phospho-Tyr residues serve as docking sites for proteins with domains that binds phosphotyrosine
what are proteins with domains that bind phosphotyrosine?
SH2 and PTB
what sort of receptor is the insulin receptor?
a receptor tyrosine kinase
how does the insulin receptor work?
starts out as a dimer. insulin binding rearranges dimer so that the intracellular tyrosine kinase is activated to cause transphosphorylation of the intracellular domains
once the insulin receptor is activated what happens?
IRS-1 is recruited to phosphotyrosine by its PTB domain, IRS-1 protein then further phosphorylated to provide docking sites for other proteins. PI3K then phosphorylates PIP2 at the 3-position to give PIP3 which becomes scaffold around which other proteins are recruited. PIP3 recruits 2 proteins to PM: Akt2 and PDK1.
how are Akt2 and PDK1 recruited to the plasma membrane?
their ‘bolt on’ PH domains which bind selectively to PIP3 and phosphorylate Ser and Thr residues
what do Akt2 and PDK1 do?
they are protein kinases that phosphorylate Ser and Thr residues. once PDK1 recruited to PM it is activated, phosphorylates Akt2
what happens after PDK1 phosphorylates Akt2?
Akt2 is further phosphorylated by mTORC2 to activate its kinase activity. Akt2 then phosphorylates substances to stimulate glycogen synthesis, and inhibit transcription of genes encoding proteins that synthesis glucose, and insert GluT4 r into PM to facilitate glucose uptake
when is mTORC2 activated and when is Akt2 activity maximal?
mTORC2 is only active in fed state, maximal Akt2 activity occurs in fed cells stimulated with insulin
what are the 2 major classes of allosteric receptors?
receptor tyrosine kinases and GPCRs
how do GPCRs work?
when activated catalyse activation of G-proteins by causing them to release the GDP they have bound in inactive state and replace it with GTP- active (GTP-bound) G protein then signals onwards
how does glucagon work?
glucagon receptor is a GPCR, stimulates formation of cAMP
what is the ‘co-receptor’ expressed on T lymphocytes that is used to recognise coat proteins?
combined chemokine receptor (GPCR) and CD4 receptor
how does Maraviroc work?
binds to recognition site of CCR5 chemokine receptor preventing it from binding HIV coat proteins so preventing HIV from infecting T cells
how does vasopressin control water reabsorption in the kidney?
binds to the V2 receptor (GPCR), stimulates formation of cAMP-> insertion of aquaporins into PM of collecting tubules
what causes congenital nephrogenic diabetes insipidus?
loss of function mutations in V2 receptor/mutations in aquaporins
how does TSH stimulate release of hormones from thyroid glands?
binds to GPCR in thyroid cell membrane
how do active GPCRs behave as exchange catalysts?
speed up the very slow dissociation of GDP from the inactive G protein by reducing activation energy of the reaction, so GTP can occupy the vacant guanine nucleotide-binding site
what condition shows the importance of slow GDP dissociation?
in boys where the subunit of Gs protein αs is mutated so GDP now only binds weakly, spontaneous activating means protein is unstable at body temperature and stable in cooler testes, leading to male precocious puberty
why is GDP dissociation slow?
if it wasn’t slow there would be no rate-limiting factor as a point of control for GPCRs
how do GPCRs provide amplification?
single GPCR in its lifetime can activate many G proteins
what terminates G protein activity?
intrinsic GTPase activity of G protein hydrolyses GTP to GDP
what can accelerate intrinsic GTPase activity of G proteins?
RGS (regulators of G protein signals) proteins
role of GPCRs in alcohol addicts (mouse models)?
stimulation of dopamine and GABAb receptors (GPCRs) signal through G protein Gi which inhibits cAMP formation. protein RGS6 enhances GTPase activity of Gi so deactivated more quickly, upregulated in alcoholic mice so more intense stimulation needed for same effect on cell. without RGS6 mice don’t become alcoholic
shared features of RTKs and GPCRs?
extracellular surface recognises the signal, transmits info to PM to activate intracellular activity, both recruit intracellular proteins that are shared with other receptors so each GPCR doesn’t require own cohort of G proteins. signal cascades are amplifying.
what are the subunits of G proteins activated by GPCRs?
3 subunits: βγ subunits are inseparable and anchored to PM. α subunits bind and hydrolyse GDP- there are 4 major families
effect of choler toxin on αs?
covalent modification to block GTPase activity. cholera bacteria restricted to gut so primary symptoms result from over production of cAMP within gut epithelia
effect of pertussis toxin on αi?
covalently modifies αi uncoupling it from GPCRs, - prevents activation
what feature of a G protein determines whether it is GTP bound (active) or GDP bound (inactive)?
presence of a single γ-phosphate on bound guanine molecule- if present then 2 conserved residues in 2 distinct domains of the α-subunit make contact with it, pull switch regions in to activate G protein and break its contact with βγ subunit. then binding of GTP to α-subunit causes the G protein to dissociate from the GPCR. once α-GTP hydrolyses the GTP the subunits reassociate
how does ACh interact with GPCRs in heart?
ACh released by parasympathetic nervous system, stimulates muscarinic GPCRs in heart that provoke coordinated response mediated by dissociated subunits of Gi that slow heart rate and reduce contraction force
GPCR general structure?
extracellular N-terminal separated by 7 TMDs from cytosolic terminal. when activated cleft opens between cytosolic ends of TMD 3, 5, 6 and 7 for the α-subunit of the G protein to insert into
how is cAMP made?
from ATP by adenyl cyclases
how is cAMP inactivated?
by phosphodiesterases
most important target of cAMP?
cAMP-dependent protein kinase (PKA)
what G protein α subunit stimulates adenyl cyclases?
αs
inhibitors of phosphodiesterases (which inhibit cAMP)?
caffein, theophylline, ventolin
what stimulates PDE3, where does this occur?
PKA (stimulated by cAMP)- in vascular smooth muscle and platelets
structure of protein kinase A?
tetramer of 2 regulatory (R) subunits (each binds 2 cAMP molecules) and 2 catalytic (C) subunits (phosphorylate substrates)
how does PKA work?
2 R (regulatory subunits), block the active sites of the 2 C (catalytic) subunits, when R binds to cAMP occluded C active site is unblocked allowing the real protein substrates to be phosphorylated on Ser and Thr residues
what are AKAPs?
scaffold proteins which associate with the R subunit of PKA to anchor PKA to different places in the cell and assemble related proteins (targets) within the PKA
similarities of cGMP to cAMP?
made by guanylyl cyclases, degraded by PDEs, many actions mediated by PKG
what is the target of Viagra?
PDE5, which degrades cGMP. prevents degradation of cGMP in blood vessels of penis causing them to dilate and blood to accumulate
PLC (phospholipase C) signaling pathway?
substrate is PIP2 (lipid from which PIP3 is made. produces 3 intracellular messengers: DAG, IP3, decrease in concentration of PIP2 is also signal.
what does DAG do after its is produced by PLC breaking down PIP2?
stays within PM, activates PKC
what are phorbol esters?
molecules which look like DAG so are tumour promoting as they activate PKC but can’t be degraded
what does IP3 do after it is produced by PLC breaking down PIP2?
water soluble so enters cytosol, stimulates Ca2+ release from the ER, Ca2+ used to regulate intracellular activities mostly through calmodulin
how is DAG inactivated?
phosphorylation
how is IP3 inactivated?
dephosphorylation
how is PIP2 resynthesised?
products of deactivation of DAG and IP3 are re-united to resynthesise PIP2
effect of Li+ on IP3 degradation?
blocks the final step (IP1 to inositol), this prevents PIP2 reformation
