Membrane Trafficking Flashcards

Question

ERAD pathway

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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.

Answer 7

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)

Answer 8

linited cell-cell contact - uninformative about multicellularity small, so high res imaging of intracellular compartments is difficult cell wall, can prevent certain studies

Answer 9

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

Answer 10

will be recessive loss of function mutants in screen so WT gene can rescue the phenotype useful for isolating the WT gene

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

-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

Answer 17

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

Answer 18

mutation of two genes results in cell death BUT the single mutations dont cause cell death suggests genes are very close

Answer 19

membrane coat formed by sec proteins drive vesicle budding from the ER these vesicles are coated by COPII

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

coated vesicles accumulate in vitro in presence of non-hydrolysable GTP

Answer 24

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

Answer 25

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

Answer 26

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?

Answer 27

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

Answer 28

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

Answer 29

COPII - forward - ER to golgi COPI - forward and backwards from golgi clathrin - later stages - incl. secretion to extracellular space

Answer 30

reversibly inhibits ARF GTPase activation golgi disassembles in minutes and fuses w ER if brefeldin washed out soon enough golgi can reform

Answer 31

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

Answer 32

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

Answer 33

Golgi Cisternae move along and mature from early to late Then COPI vesicles move backwards and transport things retrograde through golgi

Answer 34

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

Answer 35

Anti-idiotypic Ab Biochem - KDEL binding columns Genetic screens - erd mutants

Answer 36

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

Answer 37

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

Answer 38

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

Answer 39

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

Answer 40

is lethal "poisons" the golgi complex stops regular golgi activity ERD2 deletion stops golgi from working - fatal

Answer 41

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

Answer 42

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

Answer 43

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

Answer 44

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

Answer 45

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

Answer 46

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

Answer 47

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

Answer 48

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

Answer 49

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

Answer 50

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

Answer 51

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

Answer 52

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

Answer 53

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

Answer 54

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

Answer 55

-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

Answer 56

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

Answer 57

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

Answer 58

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

Answer 59

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

Answer 60

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

Answer 61

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

Answer 62

during cell division need mitochondria to segregate into BOTH daughters as they cannot be generated de novo need them to multiply and segregate them

Answer 63

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

Answer 64

regulate lipid synthesis Ca2+ signalling - ER acts as Ca2+ store mitochondrial biogenesis/fission Apoptotic signalling - signals released from MT interaction of two organelles

Answer 65

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

Answer 66

outer and inner membrane then thylakoid membranes inside in stacks for light harvesting

Answer 67

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

Answer 68

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

Answer 69

early death tissues cannot deal with free radicals and fall apart -ZSS -RCDP

Answer 70

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

Answer 71

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

Answer 72

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

Answer 73

-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

Answer 74

End- mutants that cannot internalise a fluid phase marker (eg lucifer yellow) or a bound pheremone alpha factor

Answer 75

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)

Answer 76

transferrin receptor on PM into clathrin coated pit dynamin dependent vesicle formation

Answer 77

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)

Answer 78

involves multivesicular bodies cell breaks down its own organelles so pathogen (eg virus) cant use them taken up and trashed in lysosome multivesicular bodies

Answer 79

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

Answer 80

inhibits v/t-SNARE interaction at the synaptic membrane

Answer 81

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