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Secretory pathway Flashcards

1
Q

what determines protein’s pathway in cell?

A

signalling peptides

an ER signal causes ribosome to move to ER and protein enters secretory pathway

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2
Q

what happens to a protein without an ER signal?

A

complete translation at cytosolic ribosomes

not to secretory pathway but to mitochondria/chloroplast/peroxisome

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3
Q

how do proteins cross membranes?

A

large so need channels/transporters and electrochemical/conc gradient to facilitate transport
and need signal for ligand-gated

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4
Q

when does protein sorting begin?

A

during or soon after translation

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5
Q

chaperone in protein sorting

A

help transport and can prevent folding so pass as straight polypeptide through channel

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6
Q

protein sorting in chloroplasts

A

multiple domains target passage with chaperons

stromal targeting domain pass where inner/outer membranes in contact then cleavage so no further signal and it folds

luminal domain enters thylakoid lumen then signal cleaved so folds

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7
Q

nuclear import of proteins

A

through nuclear pore complex with NLS which is NOT cleaved so proteins can re-enter just in case,
transport in folded state
F6-nucleoporins control core of complex - big aqeuous pore so molecules diffuse through

importin binds protein to form cargo complex and passes into nucleus, GEF turns Ran-GDP to GTP in nucleus, Ran-GTP interacts with F6 nucleoporin and conformational change releases cargo into nucleus, Ran-GTP complex with importin out nucleus, GAP hydrolyse GTP so conformational change to GDP and releases importin and can start again

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8
Q

ER function

A 
network throughout whole cell
synthesis
processing
sorting proteins
entry to endomembrane system
anchoring actin
communication between cells
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9
Q

co-translational translocation

A

targeting sequence added while still translated

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10
Q

integral membrane protein sorting

A

don’t enter ER lumen

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11
Q

diff targeting domains in secretory pathway

A

cell wall/secreted - signal peptide (SP)

plasma membrane - SP and transmembrane domain (TMD)

ER - SP and KDEL/HDEL (to ER for recycling)

vacuole - SP and vacuolar sorting signal (VSS)

tonoplast - SP and VSS and TMD

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12
Q

vesicle coat proteins function

A

important in loading of cargo to vesicles - docking for cargo receptors, receptors bind coat proteins on cytosolic face
Sar1 bound to GTP helps recruitment of coat proteins

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13
Q

how are coat proteins involved in membrane enclosing and vesicles budding off

A

polymerisation of coat proteins causes membrane to enclose and vesicle buds off

hydrolysis of GTP (on Sar1) releases coat so naked transport vesicles

so exposes additional proteins like SNARE so vesicles can fuse to membrane

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14
Q

vesicle coat protein types

A

clathrin - Golgi/PM to late endosomes

COPI - retrograde from Golgi to ER, interaction on internal space of cisternae of Golgi/ER to collect specific proteins, KDEL recognised by receptor proteins so retrograde for recycling

COPII - forward from ER

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15
Q

Golgi structure

A

top cis face
bottom trans face
cis to trans processing
proteins go through sacs associated with actin filaments

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16
Q

Golgi modifications

A

sequential modifications in each sac

sorting station - cisternae of Golgi stacks have diff environments like enzymes/morphology so diff functions

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17
Q

2 modes of transport through Golgi

A

vesicular transport model - bud off stack and sent to next one

cisternal maturation model - stacks move and change in environment as go through Golgi

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18
Q

final stage in secretory pathway

and 2 diff secretory pathway types

A

fusion with plasma membrane, 2 pathways

constitutive secretory pathway - need to be continuously secreted e.g. housekeeping

regulated secretory pathway - not ready or intended to be trafficked out yet, only released after signal like ER stress

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19
Q

translocon

A

in the ER membrane - basically a channel thing

the complex that transports nascent polypeptides with a targeting signal sequence into the interior (cisternal or lumenal) space of the endoplasmic reticulum (ER) from the cytosol

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20
Q

SRP

A

signal recognition particle
binds SP on protein on ribosome and guide ribosome to bind ER surface (SRP on ribosome binds SRP receptor on ER membrane)
facilitates docking on translocon so ribosome-polypeptide binds translocon (energy dependent) and co-translational translocation

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21
Q

soluble secreted proteins’ pathway through translocon

A

SP binds lateral hydrophobic pocket of translocon and peptide elongates through central pore,
signal cleaved by signal peptidase,
elongates and enters ER

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22
Q

ER membrane proteins’ pathway through translocon

A

same as soluble secreted proteins but passes through translocon until hydrophobic domain signals to stop so exits laterally into membrane
ribosomes continue to elongate peptide at cytosolic side,
further processing in membrane

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23
Q

4 modifications in ER

A

glycosylation
disulfide bonds
folding and subunit assembly
cleavage

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24
Q

N-linked oligosaccharides (ER mod)

A

14 residue precursor needed
majority needs glycosylation for correct folding and stability
make array of glycoproteins

precursor assembled on ER membrane, need phosphate on outside of ER, add acetylglucosamine and mannose residues to dolichol and phosphates,
flip reaction transfers it from cytosolic to internal space,
more ALG mediated reactions, residues to oligosaccharide,
once completed, OST (oligosaccharide transferase) transfers it to protein

N-linked so on asparagine residue of protein

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25
TM (tunicamycin)
blocks N-linked OS synthesis
26
ER quality control
monitor folding by addition of sugar groups
27
ER quality control of unfolded proteins
unfolded with oligosaccharide enters Calnexin Calreticulin cycle, 2 glucose removed from peptide, Calnexin/Calreticulin bind it and target for folding
28
what happens in ER if correct/incorrect folding?
correct is quickly secreted in vesicles incorrect enters Calnexin/Calreticulin cycle again so try again but need more glucose if goes round cycle too long it's susceptible to mannose degradation which changes and shortens branching structure and targets for ERAD (ER associated degradation pathway)
29
BIP chaperones (role in quality control)
proteins bind BIP to prevent spontaneous folding which brings it to Calnexin cycle Calreticulin brings protein to PDI system (in ER) which catalyses disulphide bridge formation/breakage PDI shuffles through diff conformations till find most energetically stable one then goes to vesicles if correct
30
ER stress
ER is sensory organelle so responds to stress | 1/3 of all proteins are secretory and 70% of proteins made under stress are secretory
31
diseases with malfunctioning ER
``` Parkinsons Alzheimers cancer diabetes obesity ```
32
what happens under ER stress
normal energy optimum thrown off balance because upregulation of secretory proteins causes folding capacity to be exceeded and can't cope, this leads to unfolded proteins accumulating in ER and causes swelling IRE1 and bZIP activate UPR (unfolded protein response), feedback loop cleaves proteins, to degradation pathway, and removes unfolded proteins
33
IRE1
transmembrane protein in ER normally monomer but dimerises under stress in ER lumen when there are unfolded proteins, it autophosphorylates and activates RNAse at cytosolic domains, binds double loop structures of specific mRNAs in cytosol and fits into pockets and causes unconventional splicing so mRNAs now encode TFs that upregulate chaperones like BiP
34
bZIP28 (plants)
ER stress induces cleavage transmembrane protein in ER to Golgi under stress S1P and S2P cleave bzip28 protein in Golgi normal P1 of bzip28 cleaved to N1 (shorter) after stress head of bZIP28 is a TF so goes to nucleus to regulate UPR genes
35
mammal equivalent of bZIP28
ATF6
36
biotic stress
damage from bacterial/fungi attack, triggers MTI (MAMP-triggered immunity) 1) recognised by receptors on cells - reactive oxidative bursts 2) induce MAPK activity which phosphorylates WRKY 3) WRKY activates stress genes and upregulates secretory pathway to deal with stress
37
MAMP
microbe-associated molecular patterns
38
autophagy
cytoplasmic material delivered to lysosomes inside double-membraned vesicles for degradation response to metabolic stress and maintenance of intracellular homeostasis
39
3 functions of autophagy
1) turning over cell content, under low nutrient conditions - provide AAs, nucleotides, lipids, sugars 2) removal of aggregates, damaged organelles, pathogens 3) cell differentiation and developmental remodelling
40
examples of developmental remodelling by autophagy
maturation of oocytes erythrocytes lose mitochondria homeostasis like neurones - stop aggregation
41
lysosome
end destination of autophagy and final of secretory pathway for intracellular degradation with hydrolytic enzymes delivered via secretory pathway in inactive state, activated when enters env. of lysosomes
42
3 types of autophagy
1) chaperone-mediated autophagy 2) microautophagy 3) macroautophagy
43
chaperone-mediated autophagy
Hsp70 bind KFERQ on proteins to be degraded | targets to LAMP receptors on lysosome which dimerise when bind KFERQ and proteins sent to lysosomes
44
microautophagy
sequestration of products proximal to lysosomes | invagination of membrane to engulf
45
macroautophagy (summary)
autophagosomes form (only in macro) 1) isolation membrane - phagophore double membrane starts to form 2) elongates till encapsulates target - autophagosome 3) autophagosome fuse with lysosome - autolysosome, release hydrolases into phagosome to digest 4) degraded to sugars, lipids, AAs and released back to cell
46
autophagosomes
double membrane structures encapsulate target in macroautophagy
47
subtypes of macroautophagy
xenophagy - for pathogens mitophagy - for mitochondria reticulophagy - for ER etc.
48
initiation in macroautophagy
INITIATION: PAS is the focal point in membrane where Atg proteins localise (on vacuoles in yeast or ER in mammals), cascade of events, 15 Atgs form autophagosome and fully enveloped in 6 mins hook like structure created on ER (autophagosome) then omegasome (buddig of ER membrane) 1) when not enough nutrients - deactivates TORC1 which activates ULK1/Atg1 complex which translocates to domain of ER 2) ULK1/Atg1 phosphorylates class III PI3K complex 3) exo84-containing exocyst complex regulates process 4) Atg9 vesicles interact for membrane stability 5) stress causes Bcl2 to phosphorylate and stops inhibiting Beclin1 so joins complex
49
PAS
pre-autophagosomal structure / phagosomal assembly site
50
Atg proteins
autophagy related proteins
51
nucleation in macroautophagy
PI3K synthesises PI3P which recruits other proteins like: DFCP1 promotes omegasome WIPIs in maturation of omegasome and isolation membrane
52
elongation in macroautophagy
recruit Atg conjugating system which activates LC3-PE and binds isolation membrane causing closure of autophagosome
53
formation of isolation membrane in macroautophagy
1) ULK1 bound with PI3K causes preformation of complex 2) ERGIC contributes to formation of larger membrane 3) proteins from range of sources with multiple functions contribute 4) DFCP1 starts isolation membrane
54
endocytic system involvement in macroautophagy
early endosome associated with PI3K complex late endosome important in signal fusion of lysosome with autophagosome
55
is autophagy selective or non-selective?
both non-selective: starved cell autophage cell contents selective: LC3 bind SLRs (sequestosome like proteins) likr p62 (sequestosome 1) which target ubiquitinated proteins to autophagosome, NDP52 and OPTN sequestosome 1-like receptors identify bacterial pathogens for xenophagy
56
3 ways bacteria is recognised for autophagy
1) induce starvation by using metabolites 2) tol-like receptors recognise bacteria and target vesicles for autophagy 3) xenophagy initiated by SLRs or Atgs
57
SLRs
target proteins to autophagosome ``` includes CLIR (LC3-interacting region), UBx (ubiquitin binding domains) ```
58
process of bacterial recognition
1) bacteria enters cell and stimulates cell to form parasitophorous vacuole to protect itself, inner membrane of vacuole has galactoside which binds galectin 2) recognised by NDP52 (an SLR) - the 2nd domain of the protein binds to LC3 part of autophagosome 3) optineurin activates NDP52 4) E3 ligase adds Ub to marked protein 5) other SLRs bind Ub and LC3 of autophagosome and engulf pathogen
59
proteasome vs autophagosome
small proteins to proteasome | large aggregations to autophagosome
60
defective autophagy
cellular collapse in starving cell, accumulation of SLRs like p62 can activate apoptosis, chronic infection if not autophage infections, but too much autophagy also source of energy for cancer
61
diseases from defective autophagy
plaques and alzheimers, cancer, CF, obesity, Crohns
62
PCD
programmed cell death in eukaryotes mechanism to remove unwanted/diseased cells from multicellular organism tissue maintenance, regulation of cell numbers, development, body function, make fingers, response to disease
63
types of PCD in animals
11 diff types but 1 is not under genetic control so not programmed and not really a PCD (necrosis) necrosis, apoptosis, anoikis, caspase and independent apoptosis, autophagy, WD, excitotoxicity, erythropoiesis, PLT, cornification, lens
64
necrosis
unpredictable, external trigger with non-specific pathway and no genetic program, no signal transduction, cytoplasmic swelling and rupture of PM
65
apoptosis
distinct and organised pathway with external/internal signal signalling cascade, predictable responses, no change to organelles, no swelling, no PM effects, only PM blebbing to form apoptotic bodies phagocytosed by other cells, and recycled (while in necrosis they are lost)
66
2 cell death pathways of apoptosis
extrinsic | intrinsic
67
extrinsic apoptosis (signals from other cells)
DISC (death-inducing signalling complex) association with plasma membrane of cell (complex consists of range of components) ligands bind to Fas receptors on target cells that have transmembrane death domain which forms FADD adaptor protein death effector domain binds procaspase 8 or 10 to complete DISC e.g. CD95 ligand bind CD95 on target cell to form DISC which catalyses procaspase 8 (initiator caspase) to become active which activates further caspases (effector caspases)
68
FADD adaptor protein
Fas-associated protein with death domain
69
procaspase 8
initiator caspase | linked to intrinsic pathway via BID
70
FLIPs
can inhibit DISC formation
71
intrinsic apoptosis summary
apoptosome formation, cytochrome C release, mitochondrial activity, controlled by XIAP (x-linked inhibitor of apoptosis) mitochondria membrane permeability causes cytochrome C release to cytosol which activates caspase 9 to attach to apoptosome and activates when dimerises to activate procaspases
72
apoptosome
``` propeller-like structure made of 7 identical monomers with several domains: CARD (caspase recruitment domain) NB-ARC (nucleotide oligomerization domain, hydrolyses ATP) made of Winged helix domain (brings ATP near so ATPase hydrolyse bound ATP) WD40 repeat (40AA repeats form circular B-propeller structure, cytochrome C binding) ``` activated by cytochrome C and ATP
73
2 mechanisms of MMP (mitochondrial membrane permeabilisation)
1) MOMP - mitochondrial outer membrane permeabilisation facicilated by BAK/BAX, BAX activated by BH3-only proteins (BID, BAD) from stress 2) MPT - mitochondrial permeability transition, pore complexes (PTPC) between membrane are opened by Ca/ROS/BAX/BAK and loss of membrane potential causes cytochrome C release
74
intrinsic apoptosis detailed
Apaf1 activated by cytochrome C and hydrolysis of ATP - opens it but monomer can't polymerise until ADP exchanged for ATP - permits apoptosome formation, cytochrome C activates caspase 9 on apoptosome which activates when dimerises and activates procaspases MMP (mitochondrial membrane permeabilization) activated by stress 1) ER stress causes Ca ion release to activate MMP 2) oxidative stress modulates protein channels BAK/bAX 3) link to extrinsic - caspase 8 activated by DISC also cleaves BID and activates BAK/BAX activation of BAK channels cause loss of transmembrane potential so cytochrome C bound to cardiolipin is released when cardiolipin oxidised
75
regulation of intrinsic apoptosis
``` BCL2 normally inhibit BAK/BAX truncated BID (activated) inhibits BCL2 so activates BAK/bAX ``` IAP inhibit caspases so limit activation cascade
76
plant PCD
no apoptotic bodies, diff proteins to inhibit BAK/BAX (no BCL2) no caspase but caspase-like activity: metacaspases (MCs) and vacuolar processing enzymes (vPEs) similar response to pathogens - hypersensitive reaction stops biotrophic pathogens by reactive oxygen species or necrosis, hypersensitive reaction - receptor recognition, resistance and activate death pathway some pathway overlap with animals
77
plants experiment with miRNAs
can use miRNAs to become resistant to virus ``` inoculate lower (oldest) plant leaves with virus then inoculate newer leaves - newer are resistant to same virus spreads by plasmodesmata to vasculature then other leaves so systemic ``` isolate viral RNA: shows RNA silencing so no infection so preventing viral replication small RNA homologous to viral RNA present in leaves in inoculated/systemic (antisense RNA to viral RNA)
78
significance of small RNAs (function)
protect against viral infections repress protein synthesis and regulate development genome stability (silence mobile elements) keep chromatin condensed and suppress transcription experimental tool gene therapy
79
small RNAs
small non-coding RNA (20-30 nts) highly conserved, in all eukaryotes except yeast 200 in plants, 700 in humans
80
3 types of small RNAs
miRNA siRNA - small interfering piRNA - PIWI interacting
81
how are small RNAs made?
RNA pol II (others in plants) 1) inverted duplication 2) locus containing opposing promoters 3) arabidopsis
82
inverted duplication
duplication, transcribed, region of complementarity so form loop, processed by DICER to small RNAs so miRNAs and siRNAs
83
locus containing opposing promoters
transcription sites on both strands and both transcribed so mRNA duplex and processed by DICER to shorter units
84
arabidopsis tas gene
TAS in plants transcribed proteins like argonaute (AGO) bind cut promotes addition of RDR6 (RNA dependent RNA pol 6) synthesise double stranded cut makes smaller tasiRNA (transacting siRNA)
85
miRNA production
1) RNA Pol II transcription to loop structure 2) association of DROSHA and DGCR8, make up microprocessor complex, cut legs off loop so pri-miRNA becomes pre-miRNA 3) pre-miRNA meets DICER in cytosol which cleaves near terminal loop (head) 4) RNAse II type endonuclease measure from PAZ region to DICER cleavage site where RIIID domain is, and cuts to double stranded RNA transcripts of fixed length 5) TRBD (TAR RNA-binding protein) bound as well as co-factor 6) all associated proteins have R3 domains, and RNA binds next to it on DSRBD (double stranded RNA binding domain) and brings double stranded RNA to domain/protein that will chop it 7) binds to AGO which gets rid of parental strand to get ssRNA bound to AGO (RISC structure) which carries out the activity
86
miRNA activity (RISC)
find and bind complementary RNA strands, cleave and degrade and interfere with activity to avoid infection, genome stability, silencing

MCB - Cell Bio (3 decks)

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