Proteins Post Midterm Flashcards
(180 cards)
1
Q
Cyclin
A
(Cyclin -> Cell Cycle)
It’s concentration works as the cell timer
CDK (Cyclin Dependent Kinase Binds to it, and gets activated)
Made continuously and destroyed periodically
2
Q
CDK (Cyclin Dependent Kinase)
A
Binds to Cyclin and defines the “active” molecule
CDK activity forms the tresholds that activate the DNA synthesis (S phase) and Mitosis (M phase)
3
Q
Wee1
A
Kinase that inhibits the Cyclin-CDK combo until the cell has grown enough (opp of Cdc25)
excess Wee1 = Too big of cells
Lack Wee1= Too small of cells
4
Q
Cdc25
A
Activates Cyclin-CDK initiating cell division (opp of Wee1)
Excess Cdc25 = Too small of cells
Lack Cdc25 = Too big of cells
5
Q
Cyclin-CDK activity
A
- Phosphorylate DNA replication machinery
- Breaks down the nuclear envelope (phosphorylation of lamin filaments )
basically it phosphorylates key mechanisms
6
Q
Kinesin-5
A
tetrameric kinesins that slide anti-parallel microtubules
+ end directed
When both + end are reached the microtubules are spread apart, driving the 2 spindle poles away from each other
pushes the centrosome apart
When inhibited = monopolar spindle + chromosome bouquet
7
Q
Dynein (anchored)
A
- end-directed motor protein
carries cargo towards - en on microtubule
positions the spindle
if dynein is anchored to the cortex, the microtubule is forcibly smashed into the cortex by the anchored dynein
Disruption of Dynein anchoring = aberrant spindle positioning
8
Q
Kinesin-13
A
create “poleward flux” of microtubules,
located at spindle poles
depolarizing microtubules from - end (gamma TURCs have been lost), pulling them inwards towards the pole,
bringing the chromosomes toward the poles
also present at the kinetochore where it triggers catastrophies and depolymerization which drives the chromosome movement
9
Q
APC (Anaphase Promoting Complex)
A
Poly-ubiquitinates M-Phase Cyclins,
(It ligates ubiquitin to the polypeptide chain) to then feed them to the proteasome (trash can)
gets its target-specificity and activation from binding partners
if no APC = no cyclin degradation, no progression to anaphase
10
Q
Cdc20
A
binding partner to APC
binds to APC, will then ubiquitinate various proteins (Securin) triggering anaphase transition
Inhibiting Cdc20 = WAIT for transition
Release of Cdc20 = GO!
11
Q
Cohesin
A
protein ring that physically ties the two sister chromatids together
needs to be cleaved by (Separase) for chromatids to separate
12
Q
Separase
A
cleaves Cohesin and allows for the separation of sister chromatids
kept secured by Securin (which is ubiquitinated by Cdc20-APC)
13
Q
Mad2
A
(Mitotic Arrest Defect)
exists in 2 conformations (Open and Closed)
its conversion is catalyzed by unaligned chromosomes
Open = Cannot inhibit Cdc20
Closed = Inhibits Cdc20 (Blocking the Anaphase transition)
14
Q
Mad1
A
Binds to unaligned chromosomes and serves as catalyst for Mad2 confrmation change
15
Q
p31
A
Protein in the cytoplasm
interacts with Mad1 and closed Mad2 tetramer when released from kinetochore
destroys Closed Mad2 & Mad1 complex
Destroys Closed Mad2 and Cdc20 complex
(Silences the wait signal)
16
Q
Aurora B
A
Kinase in the kinetochore
when phosphorylated, destabilizes the kinetochore attachments
When bi-directional, kinetochore gets stretched and aurora B get separated, releasing Mad1/Mad2
17
Q
Netrin
A
A signaling molecule that guides the neuron
will attract and repel some growth cones
(discovered by McGill graduate)
18
Q
Semaphorin
A
A type of Netrin (guidance cue)
Is a repelling signal for the growth cones
19
Q
CAM (Cell-adhesion molecules)
A
membrane proteins that mediate cell-cell adhesion and cell-matrix adhesions
Cell-cell = Cadherins
Cell-matrix = Integrins
Tight adhesion from many weak links (like Velcro)
Combination of Cis (lateral) and Trans (vertical) interactions
20
Q
Adapter proteins
A
what adhesion receptors recruit
Acts as a linker between receptor and cytoskeleton
can recruit intracellular molecules for signaling pathways
21
Q
Multi-adhesive matrix proteins + 1 example
A
Long and flexible components of the ECM that have many repeats
Regulates cell-matrix adhesion and thus cell shape and behavior!
ex: Laminin
22
Q
Cadherins
A
Confer species-specificity
calcium dependant
connects to the cytoskeleton through adapter proteins (connecting the cell to the extracellular matrix)
the layered recruitement of the adapter proteins eventually connect to actin filaments and ARP
23
Q
ECM (Extra cellular matrix)
A
diverse crosslinked of polymers
components:
- Collagen
- Laminin (multi-adhesive ECM protein)
- entactin
- perlecan
24
Q
Collagen IV
A
found in the ECM
triple helix structure similar to ropes
collagen fibrils (fibers) are built from propeptides that are cleaved and cross-linked
(the main problem with it is how to prevent polymerization of the fibers inside their ER (endoplasmic reticulum))
25
Integrins
cell-matrix adhesion molecule
straighten when activated
Bent-over = intracellular domains alpha helices stuck to each other
Straight= physically separated -> opens 2 binding sites (Talin and Kindlin)
cluster in focal adhesion complexes
they have 5 distinct layers and are connected to signaling pathways
26
Talin and Kindlin
proteins that bind to the b-integrin tail when straight (activated) and signal the cell that it is now stuck
27
Cell-surface receptors
receives chemical signals
ex: hormones released from the endocrine glands
ligands will bind to receptors according to chemical equilibrium
28
chemical equilibrium and physiological response
Ligands will bind to receptors according to chemical equilibrium
but the response of cells is often more sensitive than anticipated
reason: signal amplification
29
short term signals
modification of cellular metabolism, function, movement
ramp up and down quickly
not all or nothing
30
long term signals
modification of gene expression, development
there is a signal amplification!
all in or nothing
(cell division, diferenciation...)
31
endocrine signaling
endocrine glands release hormones that affect long distance targets
here is the functioning pathway:
hormone secretion into blood by endocrine gland -> goes through blood vessel -> gets out and onto distant target cells (where cell surface receptors are)
32
Adrenaline (epinephrine)
hormone that triggers short-term response
discovered twice and used as vasoconstrictive medicine
b-adrenergic receptors (family A GPCR)
33
GPCR (G Protein Coupled Receptors)
made of:
- 7 transmembrane alpha helices
- extracellular segments
- cytosolic segments
polypeptides that weave through the helices, forming a basket
acts like a GEF (Guaninne exchange factor)
when a ligand binds = change in conformation (signal to the cell that ligand is bound) in receptor -> allows the cytosolic segment to bind and activate heterotrimeric G protein
34
G proteins
trimeric (a, b, y) GTPase that transduces hormone signals
Ga and Gb have (antipathic helices)
Similar to Rho family GTPases (on and off states)
dissociates within seconds of ligand binding (GTP hydrolysis rate)
GDP bound = Off state
GTP bound = On state
35
antipathic helices
found on a and b subunits of G proteins
hydrophobic helices that, inserted in leaflet of plasma membrane
allows close G protein and GPCR proximity
36
GPCR signal transduction pathway (4 parts needed)
1) membrane imbedded receptor (7 alpha helices)
2) heterotrimeric G protein
3) membrane-bound effector protein
4) proteins for amplification and adaptation (ex; cAMP, Ca2+)
37
Steps to heterotrimeric G protein activation and deactivation (6)
hint: bouncing of Ga
1. hormone binding induces conf change of the receptor
2. conf change allows for Ga subunit to bind and activate
3. G protein becomes activated (GDP -> GTP)
4. GTP binding to Ga = dissociation from Gby and GPCR
5. hormone dissociates,
Ga binds to the effector (activates it)
6. Hydrolysis of GTP -> GDP
Ga dissociates from the effector,
reassociates with Gby
38
FRET (Förster resonance energy transfer)
allows to show dissociation through the use of fluorescent molecules impacting each other
allows us to measure the physical distance between 2 proteins
39
Adenylyl cyclase
a common effector of activated G proteins (GDP)
makes cAMP (secondary messenger)
40
cAMP
made by Adenylyl cyclase effector
secondary messenger
activates PKA (protein Kinase A)
Inactive PKA is made of catalytic subunits and regulatory subunits, when cAMP is present, it fills crevasses in the regulatory subunit which releases the catalytic subunits
41
PKA (Protein Kinase A)
activates by cAMP
directly controls the molecules of glycogen metabolism
42
signal amplification
multi-step activation
use of secondary messengers
reason for superior physiological reaction compared to chemical balance
43
RTK (Receptor Tyrosine Kinases)
receive signals from growth factors (NGF, EGF) and trigger cells to proliferate
key elements:
- extracellular domain (ligand binding site) receptor
- cytosolic segment (with tyrosine kinase activity) Kinase + activation arm
- C-terminal (with tyrosine residue to become phosphorylated by receptor's kinase)
Aberrant RTK signaling is found in all human cancers!
hyperactive = tumor grows
no RTK = no cell growth
when a ligand binds to the receptor it undergoes a dimerization
44
SH2
domains that bind to phosphorylated tyrosine residues
45
HER2
Has a kinase activity that facilitates all EGF family signaling
more HER2 = cell is more sensitive to signaling by many EGF family member
HER2 is over-expressed in 25% of breast cancer
46
GRB2
links active RTK to Sos
has:
(1) SH2 domain (binds to phosphorylated tyrosine on RTK)
(2) SH3 domains (binds to Sos, to proline-rich sequences)
47
Sos
Links Ras-GDP to GRB2 (which is linked to RTK)
acts as a GEF for Ras (catalyzes Ras-GDP to Ras-GTP)
48
Ras
small GTPase (on and off state)
most common mutation in human cancer
discovered in Rat Sarcoma virus
similar to Ga protein but does not have a GAP domain (need to recruit GAP protein to accelerate its hydrolysis rate, which is otherwise slow)
if always "on" -> tumor
active Ras triggers a kinase cascade
49
SH3
domain that binds to proline-rich peptides
creates a kink in the polypeptide chain
proline for 2 reasons:
- assumes an extended conformation
- binding specificity, fits into SH3 binding pocket
50
Raf
Phosphorylates MEK
activated by ras-GDP
which induces a change in Raf conformation, partially activating it
it gets fully activated when Ras GDP to Ras GTP hydrolysis releases Raf from Ras
released Raf forms a dimer with another which increases its kinase activity
51
MEK
kinase that phosphorylates target protein
Dual specificity
mainly phosphorylates MAP kinases in the activation loop
52
MAPK (MAP kinase)
can translocate into nucleus to phosphorylate different proteins (Transcription factors)
53
growth factors
stimulate cell proliferation and survival
NGF (Nerve Growth factor)
EFG (Epidermal Growth Factor)
Rita Levi-montalcini and Stanley Cohen (chicken egg and snake venom exp)
54
How does ligand binding to RTKs or EGF receptors convey signaling
Ligand binding induces dimerization of RTKs
causes a conformation change that brings the 2 intracellular kinase domains into close proximity
the kinase start to phosphorylate each other's activation loops (phosphorylation in trans)
this is the signal
activation of the RTKs is the start of cascade events!
55
3 ways cells know what is happening in their environment
1. Integrins, C-terminal tail separate aka physical separation (signal)
2. GPCR, 7 helices conformation change (signal) when the ligand binds
3. RTK, coming together phosphorylation in trans (signal)
56
additional signaling proteins recruited by phosphorylated RTKs
GRB2 (1 SH2 domain and 2 SH3 domains), Sos, Ras
57
what is the Kinase Cascade (just the order of role)
RTK -> GRB2 -> Sos -> Ras -> Raf -> MEK -> MAPK
58
what are the steps to the Ras/MAP kinase signal Transduction Pathway
(6 steps)
1. Ras activated by exchange of GDP for GTP (thanks to Sos acting as Ras GEF)
2. active Ras recruits bind and activate Raf
3. GTP hydrolysis leads to dissociation of Ras from Raf
4. Raf activates MEK
5. MEK activates MAPK
6. Active MAPK translocates into the nucleus and activates many transcription factors (related to proliferation)
59
Metastasis
requires cells to break connections with their neighbors so that it can round up and split chromosomes
focal adhesions are constantly remodeled by metastatic migration
60
contact inhibition
when you are bound to other cells it inhibits proliferation
61
Endoplasmic Reticulum
- site for protein and lipid synthesis
- single membrane bilayer organelle
- interconnected tubules and sheets , forming a network
- has a single internal space known as ER lumen
it is highly dynamic (constantly reorganized)
is functionally organized as:
Rough ER (sheets)
Smooth ER (tubules)
62
Smooth ER
Lipid biosynthesis
Contain ER exit sites (involved in ER-Golgi traffic)
mostly exists as tubules
63
Rough ER
protein biosynthesis
protein target and translocation into ER
mostly sheets
densely studded with ribosomes
64
microsomes
when cells are homogenized, the rough ER breaks up into small closed vesicles with ribosomes on the outside
which retain most of the biochemical properties of the intact ER, including the capability of protein translocation
65
reticulons
class of proteins
have a W-shaped structure that generates curvature
necessary for the formation of ER tubules and ER sheets
66
Atlastins
class of dynamin-like GTPases
undergo GTP-dependent oligomerization
necessary for the fusion of different ER tubules
67
CLIMP63
ER luminal spacer for sheet formation
ribosomes provide pressure to flatten the ER sheet and CLIMP63 prevents it from touching and keeps space for the lumen
68
What are the 3 proteins needed for ER shaping?
Reticulons
Atlastins
CLIMP63
69
Signal sequence
N-terminal
hydrophobic core (VERY IMPORTANT): 1 or more positively charge aa next to 16-22 hydrophobc residues
gets cleaved off by signal peptidase when after polypeptide goes through translocon
70
Signal Recognition Particle (SRP)
cyrolosolic ribonucleoprotein
transiently binds to both the ER signal sequence in a nascent protein and the large ribosomal subunit forming a large complex
then binds said complex to the ER membrane by binding to SRP receptor in the membrane
P54 domain is the one binding to the signal sequence (to its hydrophobic core)
71
SRP receptor (signal Recognition Particle)
integral protein of ER membrane
when it interacts with SRP (which binds to the signal sequence) it binds the polypeptide-ribosome-mRNA to the ER
it physically interacts with the translocon by having the 2 subunits GTP-bound which brings the complex closer to the ER
has an a and b subunit
the interaction is strengthened when the a subunit and P54 are both GTP-bound
hydrolysis to GDP destabilizes the interface = disassembly of the dimer
72
P54
key domain of the SRP (Signal Recognition Particle)
hydrophobic binding group that binds to the signal sequence (due to its hydrophobic core)
73
Signal Peptidase
once translation in the ER lumen is completed, it cleaves off the signal sequence, leaving it to be degraded
Right after going through translocon
74
What are the steps (8) to the targeting of secretory proteins into the ER lumen ? Cotranslational Translocation
1. ER signal sequence emerges from the ribosome
2. bound by a SRP
3. SRP and nascent polypeptide chain ribosome complex bind to SRP receptor (strengthened by GTP binding of SRP and its receptor)
4. complex properly docked to ER translocator
- Signal sequence opens translocator and enters
- Both P54 and a subunit of SRP receptor hydrolyze GTP to GDP so they dissociate (SRP recycles back)
5. The polypeptide chain enters and continues to elongate into the ER lumen as mRNA is translated
6. ER signal sequence is cleaved by Signal peptidase
7. entire polypeptide into the ER lumen
8. translocator closes and ribosome is released, folding of peptide starts
75
What are the 2 models for ER motility on microtubules?
1. Slide along microtubules
2. Growth on the + end
76
Na+ / K+ ATP pump and action potentials
establishes concentration gradients of ions
action potentials are controlled by the opening of Na+ channels
Na+ ions diffuse outward, opening neighboring Na+ channels
Action potentials are unidirectional
77
neurotransmitters
small chemical compounds
diffuse across the synaptic cleft
packaged into synaptic vesicles by H+ gradients
binding causes depolarization of the target cell
Ca2+ influx triggers exocytosis of neurotransmitters
78
Ca 2+ channels at axon terminals
Ca2+ influx triggers exocytosis of neurotransmitters
79
neuroreceptors
1. ligand-gated (like acetylcholine receptor)
2. GPCR
80
V-class H+ pumps
packages neurotransmitters into the synaptic vesicle
looks like ATP synthases (basically works in its reverse form)
uses ATP hydrolysis energy
let's in a H+ into the synaptic vesicle
81
SNARE complexes
docks synaptic vesicles at the plasma membrane
bundles of a-helix that hold 2 plasma membranes in close proximity to one another
the target of botox!
82
Botox (Botulinum toxin)
degrades SNARE complexes
undocks synaptic vesicles and prevents acetylcholine (muscle contraction) release
but! ur body still produces SNARE proteins so over time the botox fades
83
Synaptotagmin
Ca2+ sensor
Ca2+ influx causes membrane fusion and neurotransmitter release
Ca2+ binds to synaptotagmin which displaces complexin
84
complexin
Inhibits SNARE (which makes membranes fuse)
Synaptotagmin binds to Ca2+ -> displaces complexin -> SNARE conformation change (smashes membranes together)
85
Na+ symporters
reuptakes neurotransmitters
uses the energy of Na+ moving down its concentration gradient into the cell
antidepressants inhibit that reuptake, allowing serotonin to persist longer in the synaptic cleft
86
GTPase Dynamin
needed for synaptic vesicle recycling
collar that pinches the vesicle off
only 10% of vesicles are docked and used per firing (allows for multiple firing rounds)
also necessary for CCV pinching off donor membrane
87
Steps to the cycling of neurotransmitters and of synaptic vesicles (6 steps)
1. Import of neurotransmitter
2. Movement of vesicle to the active zone
3. vesicle docking at the plasma membrane (SNARE complex)
4. exocytosis of neurotransmitter triggered by influx of Ca2+
5. reuptake of neurotransmitter (Na+ symporters)
6. recovery of synaptic vesicles via endocytosis (GTPase Dynamin)
88
Growth cone
leads to axon formation
contain complex cytoskeletal machinery
89
immature neurite
small protrusions in early neuron formation will become dendrites or axons (growth cone)
90
problems for brain wiring (2)
1. Making the right connections
2. Getting neurons to the right place
91
ventricular zone
origin of neurons, which then migrate outward toward the cerebral cortex
92
cerebral cortex and neurons
neurons migrate radially long distances to pattern the cortex
needs guidance cues
93
microtubule role in cell migration
MT = front -> activate Rac (leading edge)
MT = back -> Inhibits Rho (stress fibers)
no MT = Active Rho (stress fibers)
94
Doublecortin DCX
mutation in DCX causes defects in neuronal migration
single amino acid mutation in microtubule-associated protein
female phenotype = excess gray matter near the cortex
male phenotype = smooth brain, early death
95
scratch assay
has shown that depolymerization of microtubules stops migration
cells wander around randomly
without MT, lamellopodia extends but has nowhere to go
96
Guidance cues
connect to Rho family GTPases
Attracts and repels growth cones
(netrin (attract and repel) Semaphorins (repel)
97
how do microtubules inhibit Rho
MT sequester and inactivate Rho-GEF
MT depolymerizes = release of Inactive Rho-GEF = activates Rho = Stress fiber formation
98
sarcomere
the basic unit of the "sliding filament theory"
tightly packed arrays of actin filaments and myosin filaments
separated by Z-disks
undergo rapid contraction upon muscle stimulation
structure is set by nebulin, titin and other capping proteins
99
Myosin (as a general protein type)
a protein that produces force (converts ATP into mechanical force)
100
Myosin II
type of myosin
forms bundles that pull actin filaments inward
the bundles contract the actin arrays (these contractile bundles are what muscles are made of)
the neck domain of myosin acts as the lever arm
101
Z disk
caps at the end of sarcomeres
102
The Crossbridge Cycle (5 steps)
it couples 1 ATP hydrolysis to 1 power stroke
1. Binds ATP, head released from actin
ATP hydrolysis puts myosin into a strained conformation
2. Hydrolysis of ATP to ADP + Pi myosin head rotates into a cocked state
myosin binds the actin filament in the ADP+Pi state
3. Myosin head binds actin filament
phosphate release relaxes the strain in the myosin head
4. "power stroke": release of phosphate and elastic energy straightens myosin, moves actin filament left
New ATP binding releases myosin from actin filament
5. ADP released, ATP bound, head released from actin
103
what causes rigor mortis
failure of myosin to detach in the absence of ATP
104
what are the 3 design challenges of muscles
1. prevent continuous contractions
2. activate contraction
3. freeze the structure of the sarcomere
105
tropomyosin
blocks the binding site for myosin on the actin filament
106
troponin
binds calcium and pulls tropomodulin out of the way
107
sarcoplasmic reticulum
calcium storing organelle
this calcium is needed for muscle contraction
108
motor neurons
transmit signals from the brain that activate muscle contractions
stimulation by motor neurons causes a rapid spike in Ca2+ muscle fibers
muscle contraction is stimulated by the presence of calcium
109
what are the 2 central requirements for cell division
1. increase your size
2. duplicate your DNA
110
what is the one thing cells can measure?
concentrations :)
111
Cdr2 and Pom1
(protein gradient measuring cell length im S.pombe)
small cells:
Cdr 2 wants to inhibit Wee1
is itself inhibited by Pom1
large cells:
Pom1 stays at the poles and is "far enough"
Cdr2 can inhibit Wee1
112
what are the 2 things that a cell has to do during mitosis?
1. segregate your chromosomes (perfectly)
2. degrade your cyclins
113
Sec61 complex
part of mammalian translocon
has 3 subunits
required for translocation
forms a channel for polypeptide chain
how it prevents leakage:
no polypeptide = a short helical peptide acts as a plug
yes polypeptide = middle of the central core has hydrophobic isoleucine residues that form a gasket
114
BiP
member of Hsp70 chaperone family
within ER lumen
peptide-binding domain and ATPase domain
bind and stabilize unfolded and partially folded proteins
works with Sec63 in post-translational translocation
provides unidirectionality by preventing backward movement when activated
BiP-ATP: not activated
+ luminal portion of Sec63 = hydrolysis = BiP-ADP
BiP-ADP= conformation change = binds to luminal portion and acts as a ratchet
115
Type I membrane proteins
ex: Growth Hormone receptor
N-terminal signal sequence (cotranslational translocation)
stop-transfer anchor sequence (STA)
N = exo
C = cyto
116
Type II membrane proteins
ex: transferrin receptor
no signal sequence (no cleavage)
signal anchor sequence (SA)
N = cyto
C= exo
+ charged residues at N-terminus
117
Type III membrane proteins
ex: cytochrome P450
no signal sequence (no cleavage)
signal anchor sequence (SA)
N = exo
C = cyto
+ charged residues at C-terminus
118
Type IV membrane proteins
Ex; GPCR
2+ membrane-spanning segments
pass through translocon cotranslationally in sequential order from which they emerge from the ribosome
the first segment engages in translocon, SRP, and SRP receptor-dependent manner
all the rest are not dependent and follow a strict alternation!
119
post-translational translocation steps
1. The N-terminal segment enters the ER lumen
Signal peptidase cleaves Signal Sequence
2. BiP-ATP + luminal portion of Sec63 = hydrolysis of BiP-ADP
BiP-ADP conformational change promotes binding to the exposed polypeptide chain
3. Sec63 near translocon, BiP activated at entering sites, BiP-ADP binding to luminal portion prevents backsliding and acts as a ratchet
4. Draw the entire polypeptide in the ER lumen
5. After a delay, ADP-> ATP release of polypeptide
6. folds in native conformation
BiP-ATP is recycled
120
Stop-transfer anchor sequence (STA)
stops the passage of the polypeptide chain through the translocon
makes it become a hydrophobic transmembrane segment in the membrane phospholipid bilayer
121
Signal-anchor sequence (SA)
ER signal sequence + membrane Anchor
hydrophobic so can easily move polypeptide laterally directly into the phospholipid bilayer
122
N-linked glycosylation
aids protein folding in ER
1. precursor is added to Asn in protein
2. First glucose is trimmed by glucosidase I
3a. Second glucose is trimmed by glucosidase II
3b. Third glucose is trimmed by glucosidase II
3c. New glucose is added back by glucosyl-transferase (UGGT)
4. A Mannose is trimmed by mannosidase
123
transmembrane domains (TMD)
or membrane-spanning segments
actual sequence of TMDs can be very different but each is 18-22 aa long
majority of aa are hydrophobic
forms alpha helices in the lipid bilayer
reported first in 1951 by Linus Pauling
124
disulfide bonds in the ER
(S-S)
are formed and rearranged by proteins in the ER Lumen
cannot be formed in cytosol due to the lack of specific enzymes
requires Protein Disulfide Isomerase (PDI) for disulfide bond formation in lumen
Bin in PDI active site can be readily transfered to protein by 2 sequential thiol-disulfide transfer reactions
rearrangement of disulfide bonds is accelerated by PDI
125
glycoproteins
proteins with attached carbohydrates
126
N-linked oligosaccharides
synthesized as a branched precursor in the ER
carbohydrate chains in glycoproteins attached to amid nitrogen of aspargine
127
preformed N-linked oligosaccharide
Glc3 Man9 (GlcNAc)2
128
dolichol phosphate
membrane-attached anchor
where the oligosaccharide precursor is assembled on
strong hydrophobic lipid embedded in the ER membrane
129
formation of oligosaccharide precursor
2 GlcNAc and 5 Man residues are added one at a time to dolichol phosphate on the cytosolic face
first residue = strong pyrophosphate link
after the 7 residues, flipped to luminal side
remaining 4 Man and 3Glc are added one at a time
130
Tunicamycin
blocks first enzyme in pathway synthesis of all N-linked oligosaccharide cells
131
what happens right after the entire precursor is transfered?
N-glycosylation
3 glycosidases remove all 3 Glucose and 1 specific mannose
132
Protein Disulfide Isomerase (PDI)
needed for disulfide bond formation in the lumen
Disulfide bonds in active PDI sites can be readily transferred to a protein by 2 sequential thiol-disulfide transfer reactions
this produces a reduced PDI
is oxidized by Ero1 and ready to be used again
helps accelerate the rearrangement of disulfide bonds
133
Ero1
ER resident protein
oxidize PDI when reduced
carries a disulfide bond that can be transferred to PDI
Ero1 becomes oxidized with molecular O2 diffused in the ER
134
Calnexin and Calreticulin
chaperone proteins
facilitate folding and assembly of proteins
bind to certain N-linked oligosaccharide ligands
after first 2 Glc are removed, third glucose is recognized by chaperone proteins *CNX and/or CRT) for folding
135
give 4 ER proteins that facilitate folding and assembly of proteins
Bip, PDI, calnexin, calreticulin
136
OS-9
recognizes trimmed glycans and dislocates
137
how are misfolded proteins in the ER lumen recognized for degradation (dislocation)
alpha-mannosidases trim N-linked carbohydrate chains
the trimmed glycans are recognized by OS-9 for dislocation
138
Sec63
works with BiP in post-translational translocation
provides unidirectionality by preventing backward movement when activated
BiP-ATP: not activated
+ luminal portion of Sec63 = hydrolysis = BiP-ADP
BiP-ADP= conformation change = binds to luminal portion and acts as a ratchet
139
COPII vesicles
mediate transport between the ER to the Golgi
requires V-SNAREs to function
formation =
Sec 12 (GEF) catalyzes Sar1 (GDP to GTP)
Sar1 GTP binds to the ER membrane
is followed by Sec23/Sec24 complex also binding
which provides a binding site for the second complex Sec13/Sec31
finally, Sec16 gets added to the coat
140
COPI vesicle
retrograde transport between Golgi cisternae (cis-golgi and rough ER)
mediates the transport of ER-resident luminal proteins soluble proteins back to the ER, which prevents their depletion
141
di-acidic sorting signal
sorting signal that binds to the Sec24 complex of COPII vesicles
can be found on the cytosolic segments of proteins that need to be transported to the Golgi
142
KDEL signal sequence
in most soluble ER-resident luminal proteins at C-terminus
necessary and sufficient for carrying protein to be located in the ER
143
KKXX signal sequence
On KDEL receptors and other membrane proteins transported back to ER from Golgi
at C-terminal
binds to COPIa and b subunits
necessary and sufficient to incorporate membrane proteins into COPI vesicles for retrograde transport to the ER
144
Clathrin and AP-coated proteins
clathrin coat (outer)
AP complexes (Inner)
145
Sec23-24
first complex to bind to ER membrane during COPII formation
where di-acidic sorting signals will bind
146
Sec 13-31
second complex to bind during COPII formation
147
Cargo selection
Adapter Proteins (AP) determine which cargo proteins are specifically included in a budding transport vesicle by binding to the cytosolic face of membrane proteins.
148
ARF
initiates coat assembly on donor membrane
also initiates assembly of COPI coat
149
ARF1
initiates COPI formation from Golgi
150
ARF6
Initiates CCV vesicle formation from the TGN or the PM
151
Sar1
is catalyzed by Sec12 (GEF) into Sar1-GTP which binds it the the ER membrane
and then allows for complex binding for COPII formation
152
Rab
used by cells to maintain identity of uncoated vesicles
exists in Rab-GDP in cytosol
when required it is targeted and attached to a vesicle via its liquid anchor
a specific GEF on the vesicle turns it on Rab-GTP thus tagging the vesicle
Active Rab-GTP recognizes: 1. Microtubule motor proteins 2. tethering factors
153
lipid anchor
what permits the Rab proteins to be attached to vesicles when they are needed
154
Rab effectors (2 types)
Microtubule motor proteins
tethering factors
155
Microtubule motor proteins (Rab effector)
recognized by active Rab-GTP
to propel the vesicle toward the target membrane
156
Tethering factors (Rab effector)
recognized by active Rab-GTP
on the target membrane to tether and dock the vesicle onto the target membrane
157
trans-SNARE complex
v-SNARE + t-SNARE
bring two membranes close enough together so they eventually fuse
role in
regulated secretion:
the complex is formed but does not undergo a tight wrap-around before the signal coming
constitutive secretion:
complex undergoes an immediate wrap-around after being formed, so the fusion starts right after the complex is formed
158
constitutive secretion
1. operates continuously in all cells
2. proteins without sorting signals can enter the pathway non-selectively
3. cargoes are released without external stimuli
4. a housekeeping pathway
159
Regulated secretion
1. operates only in some cells
2. proteins with proper sorting signals are selectively packaged into secretory vesicles in TGN and are often further matured/ concentrated on the way to the plasma membrane
3. cargoes are only released when cells are stimulated to do so
4. used to secret products on high demand e.g. Insulin secretion in pancreatic cells, neurotransmitter secretion in neurons
160
Maturation and concentration of insulin in secretory vesicles
proinsulin has 3 domains (B, C, A)
B and A domains are linked by disulfide bonds
close to the TGN: proinsulin is scattered, becoming more dense as it makes its way to the plasma membrane, where it matures (gets the C domain cleaved off) into insulin
161
Insulin secretion pathway
1. insulin matured and concentrated in secretory vesicles, they are docked at the plasma membrane of pancreatic cells (trans-SNARE formed)
2a. (Low blood sugar)
secretory vesicles will not fuse, no insulin release
(trans-SNARE not tightly wrapped around)
2b. (High blood sugar)
secretory vesicles rapidly fuse to the plasma membrane, large amount of insulin is released in a short time
(trans-SNARE tightly wrapped around)
162
what are 2 unsolved issues in protein secretion to the cell surface?
1. the biochemical identity of vesicles is still unknown
2. sorting signals in the trans-Golgi network in regulated secretion is unknown
163
clathrin
triskelion-shaped protein (3 heavy chains and 3 light chains)
transports protein to the lysosome via the late endosome
164
assembly and pinching off CCVs
1. CCV formation requires polymerization of clathrins and Adapter Protein (AP) on the surface of either TGN or PM
2. In TGN = AP1 interacts with integral membrane cargoes and/or cargo receptor proteins actively packaging them into CCVs
in PM = it's AP2 that selects cargoes
3. For CCV pinching off = requires Dynamin, a large GTPase undergoing GTP-dependent polymerization over the CCV neck. GTP hydrolysis releases the Dynamin
if non-hydrolyzable GTP = vesicles cannot be pinched off
165
M6P
sorting signal that allows lysosomal enzymes to be packaged into CCVs
(Mannose-6-phosphate)
M6P is only added to glycans of lysosomal proteins thanks to a sequence patch found on all lysosomal enzymes
CCVs have M6P receptors
when entering late endosome a phosphatase removes it and M6P gets recylcled back to TGN or PM
166
How is M6P added to a glycan of lysosomal enzymes?
1. glycosylated lysosomal enzyme enters cis-Golgi
GlcNAc phosphotransferase recognizes it
and adds phosphorylated GlcNAc group to terminal Mannose
2. the modified protein is released from the GlcNAc phosphotransferase
and passes to trans-Golgi
phosphodiesterase removes the GlcNAc group, leaving a phosphate at the terminal sequence
167
autophagy
a self-degradative process found in all eukaryotic cells
Dr Ohsumi discovered autophagy regulatory genes by genetic screening
has 2 levels : basal or induced
168
Roles of autophagy
1. clearance of cytoplasm (removal and turnover)
2. Nutrient recycling (amino acids, energy source)
169
what is the general process in autophagy
Induction and nucleation (Omegasome + phagophore)
expansion/ maturation (Phagosome into Autophagosome)
Fusion (autophagosome + lysosome = autolysosome)
Degradation/recycling
170
atg5
171
atg13
172
atg8 (LC3)
173
what are the two supplies for phagophore expansion?
1. Atg9 vesicles
2. Atg 18 and Atg2 channels between phagophore and ER
174
Atg18
175
Autophagy and birth example
176
177
KDEL receptor
acts mainly to retrieve soluble proteins containing the KDEL sorting signal that have escaped to the cis-Golgi network and return them to the ER
bind more tightly to its ligand at low pH, since the pH is higher in ER it allows for the release
178
how is protein transport linked to cisternal maturation?
1. COPII vesicles carrying cargo bud off from the ER, fuse one another and become CGN and Cis-Golgi
2. A newly formed cis-golgi moves and matures into the medial and the trans-Golgi and TGN. The cargoes do not leave the cisternae
3. when a cis-Golgi matures into medial-golgi, a newly cis-golgi will form just behind it
4. at TGN, the cisterna is eventually fragmented into different vesicles, which will be replaced by another TGN matured just behind it
5. COPI vesicles are responsible for retrograde Golgi transport that carry resident Golgi enzymes/proteins back to earlier Golgi cisterna
179
what are the 5 "options" of vesicle-mediated protein trafficking from the TGN
1. Retrograde transport (COPI)
2. lysosome or lumen via late endosome (AP complex+ Clathrin coated)
3. Direct lysosome (AP3 complex)
4. constitutive
5. regulated
180
Catenins
adapter proteins that allow the cadherins to connect to the actin filaments inside the cells
