Membrane Trafficking Flashcards
benefits of compartments and membrane trafficking
allows for:
-specialisation and complexity within cell
-enzymes modifying specific sub-sets of proteins in certain environments (glycosylation, proteolysis…), certain post translational machinery only found in certain compartments
-Proteins need to be moved between organelles in sequential organelles
-Retrieval and recycling of proteins/lipids back to resident compartment (HOMEOSTASIS)
downsides of compartmentalisation
cargo and trafficking factors need to get through membrane barriers
components need efficient targeting to correct compartments
need to maintain homeostasis to maintain function of the pathways
methods for studying membrane trafficking
cell biology (microscopy)
biochemistry (in vitro reconstitution)
genetics (yeast)
combining these approaches can be v powerful
George Palade expriment to describe exocrine pancreatic acinar cells
slice of guinea pig pancreas
PULSE CHASE experiment
-incubate slice in dish w radioactive leucine, gets incorporated into proteins, in this cell type most of which go through secretory pathway
-can detect where radioactivity is with photographic emulsion
-3 mins after pulse, radioactivity found all over ER membranes - where newly synthesised proteins are found
-10 mins after pulse - no longer any in ER, instead enriched in golgi
-40 mins after pulse - now localised in excretory vesicles moving out of cell
when do proteins enter the ER
proteins destined for the ER lumen are localised there co-translationally (ribosomes dock on the Rough ER)
GFP pulse chase (more modern method for George Palade experiment similar)
instead of radaioactivity
use GFP tags and Fluorescence microscopy
can use drug that halts protein synthesis to end the Pulse
What does crossing of compartment membranes require?
requires:
-signal sequence
-sometimes an RNP signal recognition particle (SRP)
-signal receptor
-translocation channel
-source of energy, ensuring unidirectional transfer
Bloebel’s in vitro reconstitution of ER translocation
mix fractions from different cell types in vitro to reconstiture complicated biochemical reactions in vitro
eg translocation across ER
Break up ER into vesicles
Microsomes constructed from rough ER have ribosomes so are more dense
allows fractionation to isolate them from other membranes in the cell
Any labelled protein synthesised and translocated by these will be protected within the microsome from added proteases
(control where detergent is used to disrupt the microsomes)
2 types of ER import
-Co-translational
-Post translational
depends on the type of protein
in co-translational - completed proteins are unable to cross the membrane due to their folding - even if they have the signal sequence
-NEEDS to be done during synthesis before folding
-would need to completely unfold them to get them across translocon
i guess can test this by either conudcting the microsome/protease experiment during translation (adding IgG mRNA) vs adding fully translated protein (eg IgG)
Co-translational translocation process
-Ribosomes on the mRNA
-Translation produces N-terminus first - where the signal sequence is (10-12 hydrophobic AAs)
-An SRP (signal recognition particle) recognises the signal sequence as it exits the ribosome
-SRP is recognised by ER membrane receptor
-GTP hydrolysis (by the receptor?) releases the SRP and Flips the Signal peptide into the channel
-translocation as rest of polypeptide is pushed through into ER lumen
-SRP can be recycled for another round of signal recognition
-secretory protein in the ER lumen has its signal peptide cleaved by Signal peptidase
protein can now be Folded
The SRP - signal recognition particle
Highly conserved
bacterial form can target mammalian proteins to ER lumen
an RNP(ribonucleoprotein) made of folded RNA and protein
Problems with identifying ER import channel
is a hydrophobic membrane protein
hard to study in lab
Experiment for studying the ER translocation channel
cross linking experiment
Photoreactive Lysine residues engineered into protein that crossed ER membrane (Preprolactin)
cross link with light
i guess can then immunoprecipitate via the known peptide
identified the Sec61 polypeptide - oligomerises to form the channel
lipid vesicles containing Sec61 sufficient for translocartion
energy source for co-translational translocation into ER
chain elongation at the ribosome
the energy of peptides being added pushes it through
Yeast Sec mutant selection
so that only mutants defective in the Sec pathway will grow
use histidine production enzyme as reporter:
-add ER translocation tag
-WT cell translocate into ER where Histidinol isnt present - so cannot produce histidine
-No such translocation in Sec mutant: reporter enzyme remains where it is and can produce histidine
grow on medium w/out histidine but with histidinol
Post translational translocation into ER lumen: signal peptide difference
Have signal sequences that are not sufficiently hydrophobic to engage the SRP until AFTER translation is complete
post translational translocation into ER requirements
requires:
-Cytosolic components
-ATP (no energy from peptide chain elongation by ribosome)
-use sec61 channe, but assisted by additional membrane factors (sec62/63)
-ER resident protein Kar2/BiP/GRP78 acts as molecular ratchet to create force for translocation
Cytosolic components of Post translational translocation
HSP70 chaperones maintain the substrate in a translocation competent state
Post translational translocation into ER lumen process
– signal peptide associates w SRP, associates w receptor
– polypeptide/chaperone (HSP70) complex associates with translocon (Sec61)
– Sec62/63 complex next to channel
– luminal binding protein (BiP) binds the protein on the lumen side
– hydrolyses ATP and ratchets the polypeptide into lumen
what happens inside the ER?
-signal peptide cleavage
-glycosylation (cell:cell adhesion, communication)
-Folding (disuflide isomerase eg)
-Further proteolytic cleavage (mainly happens in golgi tho)
-quality control
ER lumen - Addition and processing of N-linked oligosaccharides
-polypeptide enters ER lumen
-carbohydrate chains transferred by donor ASAP
-N-acetyl glucosamine, with mannose on it, then glucose on the end of that - all in 1 step
-N-linked oligosaccharides typically added to Asparagine residue (typically Asparagine-x-Serine-Threonine)
the glucose and one of the mannose is trimmed off before transfer to golgi
Quality control in the ER lumen: Calnexin and Glucosyl transferase
-unfolded protein - has glucose on its n-linked oligosaccharide
-glucose trimmed, partial folding
-different form of carbohydrate on it now - recognised by Calnexin (membrane protein), which hangs on to it by the single glucose on end
-it is then recognised by glucosidase, cleaves this glucose so calnexin not holding anymore
-
-if properly folded can leave
-if not, but is in conformation that glucosyl transferase recognises - then it adds back the glucose
-can be held by calnexin again
-this keeps it in the ER until glucosyl transferase no longer recognises it (presumably when all the hydrophobic residues have folded to face inwards) and it is properly folded
-can exit ER after that
formation and rearrangement of disulfide bonds in ER lumen:
PDI protein can change electrons and enable various forms of disulfide bonds to be tested
PDI itself needs to be oxidised to carry these out
-Ero1 protein helps in this
-Ero1 itsefl oxidised by free oxygen diffusing into ER
can rearrange incorrect disulfides until best conformation reached
Misfolding in the ER - Export and degradation purpose
sometimes misfolding is so bad that machinery cannot sort it out to give functional fold
-dont want to keep passing this useless protein around, or out of the cell and lose resources
-instead cell recycles the AAs
ERAD pathway
ERAD pathway
ER associated degradation pathway
-misfolded proteins recognised by resident ER chaperones
-spending too long in a complex with these ends up with being passed through a translocator out of ER to cytoplasm
-Translocator has AAA-ATPase - helps force the polypeptide out
-cytoplasmic polyubiquitin adds ubiquitin chains onto the polypeptide as it enters the cytosol
-Directs it to proteasome where it is degraded into its constituent AAs
Unfolded protein response summary
Normal proteins dont leave ER until properly folded
mutations causing misfolding block exit from ER
proteins remain bound to folding chaperones
eg:
-BiP
-Calnexin
cells respond to the presence of these unfolded proteins by increasing transcription of chaperone genes
need signals to get from ER to nucleus
involves a number of pathways:
-IRE1 pathway
-PERK pathway
-ATF6 pathway
IRE1 pathway
a Splicing pathway
misfolded protein recognised by ER
-IRE1 Transmembrane Protein kinases act as misfolded protein receptors
-recognition causes them to dimerise
-unmasks their endoribonuclease activity
-can now splice out a key intron in a particular pre-mRNA
-this mRNA now produces a TF
-turns on chaperone genes in nucleus
-extra reinforcements helps deal with misfolded proteins
PERK pathway
PERK TM receptor kinase
-kinase activity phosphorylates and inactivate a Translation initiation factor
-reduces the levels of new proteins translated - reducing proteins trying to enter ER which could cause more problems (eg blocking pathways)
also allows for selective transaltion of the Chaperones
AFT6 pathway
Receptor undergoes conformational change when activated
leads to its regulated proteolysis in the Golgi
releases a subunit which acts as a TF for activating chaperones
part of multiple TFs that go to nucleus turning on chaperones to increase folding capacity of ER in response to stress
Example of protein folding complications in cell: Cystic Fibrosis - CFTR F508 deletion
CFTR - cystic fibrosis TM conductance regulator
takes 30mins to translate and fold
-even in the WT a significant fraction is targeted for degradation
the F508 deletion causes nearly the entire pool to not fold well and be degraded
destroying the entire CFTR pool before it can reach the membrane
the delta-F508 mutant can actually function if it reaches the membrane
but it never does as the mutation causes it to be recognised as a target for degradation.
Advantages of yeast as model organism
can grow as 1n and 2n, good for genetic studies
entire genome known and annotated
cheap and easy to grow in large quantities - good for biochem studies
conserved fundamental pathways (eg secretory)
disadvantages of yeast as model organism
linited cell-cell contact - uninformative about multicellularity
small, so high res imaging of intracellular compartments is difficult
cell wall, can prevent certain studies
screening genes required for secretion pathway
whole pathway too complicated to reconstitute as one in vitro
so use genetics
so instead isolate mutants defective in secretion
-is essential process so use temperature sensitive/conditional mutants
mutant will be defective in membrane trafficking
rescue experiments in yeast secretion mutants
will be recessive loss of function mutants in screen
so WT gene can rescue the phenotype
useful for isolating the WT gene
identifying secretion defective mutants
vesicles are prevented from being secreted
so secreted proteins in the vesicles accumulate within the cell
leads to increase in cell density
-can mutagenise yeast cells
-centrifuge/fractionate these sec- mutants out from others at restrictive temperature
the accumulated proteins would be normally secreted ones
-invertase
-acid phosphatase
analysing the sec- mutants collected in the density screen
can look at alterations in normal ultra-structure of cells with Electron Microscopy
eg:
-accumulation of vesicles
-abberant membranous structures
-accumulation of golgi discs stacking together
certain proteins can also be detected that are modified at diff stages in the secretory pathway (glycosylation, proteolysis)
eg look at ability to secrete invertase and acid phosphatase permissive/restrictive temps
-sec- mutants fail to export active versions of these BUT continue to synthesise protein under restrictive temps
identifying genes from sec mutant screens
complementation tests
-cross two mutants
-if phenotype is not rescued then can assume that mutation is in same gene
-if rescued then in diff genes
identifying order of genes in the secretion pathway in the sec mutants
eg sec7,18 double mutant
– use invertase as marker,as it simply gets bigger as it goes through the secretory pathway
– all mutants arrest at some point in the secretory pathway
– but diff mutants at diff points - where different carbohydrate modifications are present on invertase
run invertase from sec7, sec18, sec7,18 and sec18, +known later mutation on gel
the double mutant invertase looks like the sec18 single mutant on gel
so sec7 is important LATER in the pathway than 18
yeast alpha factor different modiifications through sec pathway
WT secreted alpha factor is cleaved into multiple small peptides
if arrested before that will go ER-Golgi-Vacuole and be one large protein instead
can see diff in size on gel
different classes of Sec mutants
-class A: ER import defect
-class B: Stuck in ER, ER budding vesicles not formed
-class C: Stuck in ER to golgi transport vesicles
-class D: accumulation in golgi - defective in transoirt from golgi to secretory vesicles
-class E - accumulate in secretory vesicles, transport from vesicles to cell surface defective
downside of yeast mutagenesis screen - not capturing all secretory pathway genes
the temp sensitive screen identified ts sec mutants, not all genes can cause this phenotype when mutated
then only considered secretion to plasma membrane
defects in transport to vacuoles, endosomes not idetified
any redundantly functioning genes wouldnt be identified
relevant due to historic WGD in S. Cerevisiae
synthetic lethality
mutation of two genes results in cell death
BUT the single mutations dont cause cell death
suggests genes are very close