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
COPII
membrane coat formed by sec proteins
drive vesicle budding from the ER
these vesicles are coated by COPII
assembly of COPII coats
Sar1 drives assembly of COPII coats
binds Sec12 on ER membrane on the cytosolic side
Hydrolyses its bound GTP, causing a conformational change where its hydrophobic N-terminus becomes lodged in the membrane
Sec23/24 bind Sar1 and cargo on the cytosolic side of membrane and form the COPII coat
Sec24 activity in COPII coat formation
is a coincidence receptor
binds cargo proteins and Erv14 (cargo receptor) to drive ER export
interacts with cargo in the vesicle
this communicates cargo density
so vesicles can coordinate budding when a good amount of cargo is present
Jim Rothman’s cell free assay of golgi transport stuff
viral protein from VSV-infected WT and mutant cells
can use modifications present on this protein to monitor where in golgi it is
mutant cell’s viral protein cannot process these carbohydrates in its golgi as the membranes dont have the correct enzyme
so is transported w/out modification
But incubate this protein isolated from the mutant cells
with Golgi isolated from WT non infected cells
protein can now be modified
can use these modifications as transport location markers
vesicles and non-hydrolysable GTP
coated vesicles accumulate in vitro in presence of non-hydrolysable GTP
COPI coated vesicles
was first assumed that these vesicles were transporting cargo through the golgi
ARF GTPase dimerisation important in assembly
are they secretory or retrograde
or both
SNARE hypothesis
Each vesicle has one kind of SNARE on it (V SNARE)
its target membrane has a diff kind of SNARE (T SNARE)
-will drive the fusion reaction between these membranes - also drives specificity of membrane fusion (along w other specificity factors)
SNAP receptors also in membranes
zippering (SNAREpins) drives fusion
Rab GTPases at further specificity
V SNARE not involved in budding
but once budded off, SNAREs are key drivers of vesicle and target interaction and fusion
SNARE specificity and evidence
used artificial liposomes to test all potential yeast v-SNARES for their capacity to trigger fusion by partnering with t-SNARES for:
-golgi membrane
-vacuole membrane
-PM
which v-SNAREs like to fuse with which t-SNAREs?
Vesicle fusion process
Vesicle has v-SNARE and Rab-GTP on surface
Rab-GTP interacts with Golgin (tethering factor)
-docking step, interaction with surface but no fusion yet
This allows v- and t-SNAREs to interact
forms trans-SNARE complex
GTP hydrolysis on Rab-GTP->GDP during fusion via trans-SNARE complex
forward transport signals in sec pathway
there is specific uptake of secretory proteins and v-SNAREs into COPII-coated vesicles
both Sec23/24 COPII coat proteins are necessary to recognise cargo-packaging signals
Erv29 (conserved membrane protein) required to package pro-alpha factor into COPII vesicles
Export is saturable and depends on level of Erv29 expressed in cell
COPII vs COPI vs clathrin vesicles
COPII - forward - ER to golgi
COPI - forward and backwards from golgi
clathrin - later stages - incl. secretion to extracellular space
Drug experiments - Brefeldin A
reversibly inhibits ARF GTPase activation
golgi disassembles in minutes and fuses w ER
if brefeldin washed out soon enough golgi can reform
Golgi Cisternal Progression
whole stacks of Golgi moving and maturing
(as opposed to backward transport through golgi via transport COPI vesicles)
eg algal scales
-too large to fit in transport vesicles
-same case for collagen aggregates in humans
Live imaging evidence of cisternal progression
Vrg4-GFP - expressed in early golgi - green
Sec7-DsRed - late golgi expressed
stacks of golgi are dynamic - are present throughout cell not stacked together
yet still get sequential transport
focus on one green spot (early golgi) and track over time
-starts green
-becomes yellow (coexpression GFP+DsRed fusion proteins)
-Then red, losing early marker
can argue that this shows it maturing over time
Model of Cisternal Progression
Golgi Cisternae move along and mature from early to late
Then COPI vesicles move backwards and transport things retrograde through golgi
proteins w/ KDEL signal
ER resident proteins (PDI, BiP, GRP94) have c-terminal KDEL sequence
this is necessary and sufficient for ER localisation
HOWEVER their Carbohydrate modifications show that they reach the Cis-Golgi
so they are retrieved/recycled BACK INTO the ER
-reach golgi and come back to ER
-its a waste to secrete these ER proteins that get caught up in COPII coated vesicles
-so mechanisms to retrieve/recycle them
-also for recycling membrane too
Identifying the receptor for KDEL sequence
Anti-idiotypic Ab
Biochem - KDEL binding columns
Genetic screens - erd mutants
Anti-idiotypic Ab (identification of KDEL receptor)
2 cycles of immunisation:
-inject KDEL into mice
-get anti KDEL Ab
-inject these anti KDEL Ab into another mouse
-get anti-anti-KDEL Ab
Hopefully get some of these Anti-anti-KDEL Ab which interact w the KDEL receptor in the same way KDEL does
-screen cell extracts for proteins that they interact with
-in hope to find KDEL receptor
this identified a 72kDa protein but it did not have the right properties
risky apprach
lots that can go wrong
Biochem - KDEL binding column
good chance that KDEL receptor is a membrane protein
hard to work with biochemically
also dont want KDEL binding the receptor in the ER but in golgi condiitons
so binding assays may have wrong conditions for binding and hence not find it
Genetic screens for erd mutants
mutagenise yeast
check for ER resident protein recycling defects
Invertase-FEHDEL fusion protein
-only 5% is secreted in non erd mutants
-but is golgi modified
-so is recycled
use colony assays to detect external invertase activity shown in recycling mutants
complementation tests identified erd1 and erd2
-their protein products are involved in either HDEL recognition or in the recycling process
-can sequence the genes and infer their function through sequence
ERD1 is golgi membrane protein
ERD2
encoded the HDEL receptor
overexpression of it increases the capacity of the HDEL recycling system
suggesting it is the receptor
determine the specificity of the sorting system
eg Kluyveromyces lactis ERD2 recycles DDEL when expressed in S. cerevisiae
-also suggesting receptor activity
ERD2 deletion mutant
is lethal
“poisons” the golgi complex
stops regular golgi activity
ERD2 deletion stops golgi from working - fatal
Golgi to ER retrieval
ER proteins end up in COPII coated vesicle
transports them to golgi
KDEL receptor recognises the KDEL peptide on the ER protein
retrieves them and recycles them back to the ER in COPI coated vesicles
Recycling of membrane proteins
similar idea
diff signals to luminal ER proteins
-KKXX cytoplasmic c-terminal retrieval signal
-XRRX signal
COPI mutants are unable to retrieve these
and so is important in their retrieval
Trans Golgi sorting
some proteins go straight to PM for secretion
some to endosomes
some to lysosomes
and some back to ER
several mechanisms to sort them
diff vesicles have diff coats
eg:
-COPI
-Clathrin
two pathways to cell surface:
-Regulated one
-Constitutive one
The Exocyst (eeyikes!)
Octameric complex of Sec proteins
invovled in tethering secretory vesicles to the PM prior to SNARE mediated fusion
important for final exocytosis step
eg for a vesicle with PM targeted proteins
cell cycle regulated
-because is also involved in targeting things to cytokinetic ring in cell division
Regulated vs constitutive secretion
all cells constitutively secrete
but some are also able to store proteins in special secretory vesicles
-can sort proteins - no known clear signals tho
-Can aggragate (with Chromogranins) due to the lower pH of the late golgi
proteases process these secretory proteins
process pre-forms in to mature secreted forms
eg:
-Pro-insulin in earlier less dense vesicles
-cleavage in early vesicles
-mature insulin in later mature vesicles
Clathrin coats:
Heavy chains and light chains
forms triskeleton shape that comes together to form coat
are present on the cytoplasmic side of membrane
start of as coated pits that help form vesicle
ARF GTPases found in these coats - initiate coat assembly
mediate transport
T-Golgi to endosome
PM to endosome
Golgi to lysosome
Adaptor proteins - specificity in golgi sorting
adapter protein complexes
4 subunits
fill space between clathrin lattice and the membrane
bind cytosolic face of membrane proteins
sort cargo
AP1-endosomes
AP2-PM
AP3-lysosome
GGA-to endosomes
Dynamin
Performs the final step of vesicle pinching off and budding
requires a bit of energy
forms ring around budding vesicle neck
interact with clathrin and membrane proteins
Dynamin hydrolyses GTP and uses the energy to pinch off
not requried for COPII or COPI vesicles
-dimerisation of the ARF GTPase may substitute it
Vacuolar/Lysosomal protein sorting
Lysosome/vacuole full of proteolytic enzymes - need to keep them separated from rest of cell in there
so mis-sorting them can be bad
functions to degrade extracellular material taken up by endocytosis and also some intracellular components
lysosome resident enzymes are transported there through secretory pathway
at late golgi (Trans golgi network) they are sorted into pathway destined for lysosomes rather than PM
modification with Mannose-6-phosphate important for targeting
Lysosomal storage diseases
I cell disease: large inclusion bodies in lysosomes
the lysosomal enzymes are secreted instead of targeted to lysosome
cells lack the N-acetyl glucosamine phosphotransferase (no M6P on the residue on their proteins - a targeting signal)
BUT can take up M6P from cell surface so can still recognise M6P - have the receptor
just cant modify own proteins
Mannose-6-Phosphate and M6P receptor
Modification that targets soluble proteins to lysosomes
recognised in Trans-golgi network (pH6.5) by M6P receptors
M6P receptors are sorted into clathrin/AP1 vesicles
Bud, lose coats, then fuse with late endosome where cargo is released
-coat needs to be removed to expose SNAREs and reveal specificty/vesicle knows where to go
MGP receptor recycled
endosome fuses with lysosomes
same machinery also perfomrs endocytosis of M6P from outside of cell via the M6P receptor on the PM
Vacuole/Lysosome protein sorting screens
Carboxy peptidase Y protein targeted to vacuole
look for mutants that secrete it instead
look for extracellular CPY activity
can then combine mutants for complementation test/test the order of action of the genes in the pathway
sorting to late endosome - CPY pathway
Carboxy peptidase Y synthesised in prepro form
transported through ER to golgi
Sorting:
in late golgi CPY is specifically recognised by a receptor Vps10
receptor mediated sorting
Transport:
cytoplasmic factors:
Clathrin
2 adaptors Gga1, Gga2
CPY dissociates from Vps10 at late endosome and is transported to vesicles where it is cleaved to generate mature form
Vps10 is retrieved to the late golgi through specific aromatic based signal in its protein seqeuence
Sorting from golgi straight to vacuole - skipping endosome sorting
ALP and Vam3 traffick from golgi to vacuole directly bypassing endosomes
both proteins contain cytoplasmic domains with acidic dileucene sorting signals required for packaging into the correct sorting vesicles
Vesicles transporting ALP to the vacuole require AP3 but not Clathrin
Mitochondria targeting in vitro reconstitution
-mt protein with mt uptake targeting sequence in tube
-add energised mitochondria
-proteins w signal sequence will be taken up
-add trypsin to tube
-only proteins taken up will be protected from proteolysis
there is an ATP requirement to get these proteins across membrane
chaperones to unfold already folded proteins inc cytoplasm and get them across
where they refold
targeting process through both mitochonrial membranes into matrix
eg N-terminal targeting sequence that targets it all the way into matrix
-matrix signal recognised by import receptor
-receptor moves close to translocon
-signal goes through import pore followed by rest of polypeptide
-often keeps on going through different translocon on inner membrane if it has the right signal
-the Matrix chaperone Hsc70 pulls it in
-matrix processing protease cleaves the signal
-refilding of protein w chaperone help
importance of unfolding and mitochondra targeting
fuse DHFR to protein that is normally imported into mitochondria
drug molecule induces DHFR to fold into compact form
prevents fusion protein from passing all the way through
part of it goes through both the outer and inner membrane translocons byt DHFR blocks it from going all the way through
-Pull the membranes together
can count the translocation sites
along with electron microscopy and Ab to DHFR w gold particles
Energy in mitochondrial translocation
ATP hydrolysis performed by cytosolic and matrix Hsc70 (chaperone)
mitochondria also pump protons across their membranes - generates a proton motive force
not sure exactly why it is needed for import
but cyanide leads to precursors binding receptors but no import
-perhaps due to +ve charges in the amphipatic helix are “elecrophoresed” and moved into the -ve charged interior by the membrane potential
mitochondrial inner membrane translocation
not everything needs to go all the way into matrix
some are targeted to be transmembrane in the inner membrane:
start w matrix signal
then have signal along sequence that halts importthrough inner membrane
so that region stays in the membrane
-> TM protein
types of TM proteins in mitochondria inner membrane - how they establish
1 pass - 1 halt signal that stays in membrane
2 passes - import -> 1 bit stay, then export of other part - 2nd pass - both ends on same side
many passes - multiple internal sequences that cause multiple passes through the membrane
Mitochondrial intermembrane space targeting
Protein begins with matrix target sequence
then an intermembrane targeting sequence
cleavage of the intermembrane targeting sequence at the inner membrane translocon so protein ends up in IM space
OR
an intermembrane targeting sequence
goes through outer membrane
then Erv1 generates disulfide bonds
Mia40 transfers them to the protein
idk maybe not so important
guess its forcing the protein to fold before passing through inner membrane
Mitochondrial fusion and fission
during cell division
need mitochondria to segregate into BOTH daughters as they cannot be generated de novo
need them to multiply and segregate them
Inheritance of different organelles
ER and mitochiondria cannot be generated de novo
kinesin and dyenin driven movement across MTs used to move them in animal cells
ER (Myo4) and Mitochondria (Myo2) transported on actin by Myosin
ER - Mitochondria contacts
regulate lipid synthesis
Ca2+ signalling - ER acts as Ca2+ store
mitochondrial biogenesis/fission
Apoptotic signalling - signals released from MT
interaction of two organelles
Mitochondrial fission by interaction with ER
Have mitochondria and ER
kinesin motor and adapters links Mitochondria to motors
ER wraps around mitochondria
DRP (dynamin related protein) GTPase helps w fission - cinches off
Chloroplasts
outer and inner membrane
then thylakoid membranes inside in stacks for light harvesting
Peroxisomes
Can assemble de novo in eukaryotic cell
happens depending on physiological state of tissue - may or may not want them active
single membrane enclosed
diverse reactions - involving lipid metabolism
also a defense system for scavenging peroxides/ROS
PEX genes encode peroxins
which control:
– assembly
– division
– inheritance of peroxisomes
Peroxisome import
Signal peptide: PTS1 signal
-recognised by PEX5 soluble receptor in cytoplasm
-PEX5 receptor recognised by peroxisome membrane protein PEX14
-imports it along with the bound cargo protein
-cargo released and can go where it needs to
PEX5 needs to be recycled and exported from peroxisome - export channel encoded by PEX10 and 12
Disease relevance of peroxisome biogenesis disorders
early death
tissues cannot deal with free radicals and fall apart
-ZSS
-RCDP
PTS signals and receptors
PTS1 (C-terminal -SKL) recognised by Pex5
PTS2 (N-terminal nonapeptide) recognised by Pex7
PTS receptors can be recycled or Poly-Ub and degraded if peroxisomes not required
New peroxisomes generated by growth and fission of existing organelles
or by de novo biogenesis from the ER
-is the only ER derived organelle w its own Translocon
Peroxisome biogenesis from the ER
involves vesicular transport of Peroxisomal membrane proteins (PMPs) from the ER
-Pex proteins leave ER in separate vesicles (diff proteins in each)
-vesicles later fuse - Heterotypic Fusion
-This combines the different Pex proteins in the same vesicle
-This allows it to begin importing Peroxisome targeted proteins
this means that peroxisome targeted proteins wont be targeted to the ER - just into peroxisome once Pex vesicles have fused to make it
Endocytosis
PM invaginates into cell resulting in production of vesicle
that can then fuse to lysosomes
can be used to retrieve proteins that are part of secretory vesicle for recycling
downregulation of cell surface signalling
remodelling cell surface lipid/protein composition
also is a weak point for entry of pathogens and toxins
Different modes of endocytosis
-Phagocytosis - large particles, actin mediated
-pinocytosis - liquid intake, membrane ruffling
-caveolae
-transcytosis - in polarised cells, endocytosis at one end of the cell followed by exocytosis at the other end
-Receptor mediated endocytosis - recognise something at cell surface via receptor
bind it
this enriches it in coated pits
endocytosed
Screening endocytosis defective mutants in yeast
End- mutants that cannot internalise a fluid phase marker
(eg lucifer yellow)
or a bound pheremone alpha factor
Cholesterol uptake
Transported in blood as cholesterol esters in form of low density lipoprotein (LDL)
cells need this to make new membranes, so they express a cell surface TM receptor that recognises LDL at neutral pH
internalised via Clathrin coated pits
NPXY sorting signal in receptor tail that interacts with AP2
dynamin dependent formation of vesicle
GTP hydrolysis releases coat
targets to late endosome
LDL released at low pH
then to lysosome
receptor recycled to PM
mutant receptor/AP2 leads to atherosclerosis (coronary heart disease)
Transferrin Fe uptake
transferrin receptor on PM
into clathrin coated pit
dynamin dependent vesicle formation
Multivesicular bodies
eg for trashing internal structures in the lysosome (badly damaged mitochondria…)
Inward budding of the endosome membrane into the lumen
-allows for lysosomal degradation of cell surface receptors (desensitised or damaged)
cargo is tagged w ubiquitin (mono), interacts with ubiquitinated Hrs and ESCRT proteins in the invaginations (endosomal sorting complexes required for transport)
Autophagy
involves multivesicular bodies
cell breaks down its own organelles so pathogen (eg virus) cant use them
taken up and trashed in lysosome multivesicular bodies
Synaptic vesicles
similar to regular secretory vesicles
have recycling cycle
-exocytosis/secretion
-followed by endocytosis/recycling
-proton antiporters create proton gradient which drives neurotransmitter import into vesicle
-Synaptotagmin on vesicle surface is important for fusion to membrane
-targeted to membrane via v/t-SNARE interactions
-though these alone are not sufficient to drive fusion at synaptic cleft so remain docked there
-until wave of Ca2+ import
-causes fusion to membrane
botox treatment and vesicle fusion
inhibits v/t-SNARE interaction at the synaptic membrane
Calcium influx - fusion in response action potential
action potential opens up calcium channels in membrane
causes calcium influx
causes Synaptotagmin to undergo a conformational change
causes release of complexin from the v/t-SNARE complex
causes membrane fusion
<1millisecond
