Secretory Pathway Flashcards
components of endomembrane system
endosome, nuclear membrane (extension), lysosome, golgi, ER with ribosomes, plasma membrane
nuclear pores
proteins in, mRNA out outer nuclear membrane IS continuous with ER - fold around at nuclear pores, transcription factors in and mRNA can leave, NOT protein lined - continuity btwn cytoplasm and nucleus
Rough ER jobs
secreted and membrane protein biosynthesis steroid synthesis oxidative modification of xenobiotic COP II vesicle formation
Smooth ER jobs
generation of limiting membrane in autophagy
Rough and Smooth ER jobs
phospholopid and cholesterol synthesis Calcium storage (especially in muscles) MHC I ag presentation
ER client proteins
secreted proteins (enzymes, Igs, extracellular matrix proteins) integral membrane proteins of endomembrane system (receptors, transporters, channels, cell adhesion) lumenal proteins of endomembrane system (lysosomal hydrolyses, ER chaperones)
non-ER client proteins
made on free polysomes in cytoplasm and imported where they need to go cytosolic (cytosekeletal, contractile, soluble enzymes) peripheral membrane proteins on cytoplasmic face (spectrin) nuclear, mitochondrial, perixisomal proteins
translocation
protein synthesis on cytoplasmic side of ER membrane, nascent polypeptide is translocated through the protein lined channel into the lumen where modifications happen
N-linked glycosylation
HIV gp160 insulin receptor, addition of pre-assembled Glu3-man9-glcnac2 branched sugars to amide groups of Asparagine
co-translational glycosylation
the signal for glycosylation is linear! add glycosylation before polypeptide folds, enzymes transfer in ER lumen as translate - amino end of protein can be in the lumen or the cytosol, transferred over to reside
oligosaccharyl transferase
transfers phospholipid of sugars made in the ER by enzymes onto Asn of nascent polypeptide cytoplasm is a reducing environment, make it as the protein is folding, transfer from dolichol in membrane
chaperones
facilitate folding etc. in the Er
proinsulin
protein with 3 disulfide binds - needs chaperone to facilitate oxidation
BIP
chaperone binds to hydrophobic stretches of AAs in the ER i.e. proinsulin and glycoproteins bind to AAs, hydrolyze ATP, fall off, binds new stretches of hydrophobic AAs on peptide - if fold naturally, it won’t be right, BIP binds to prevent from folding in a way that can’t be unwound during/after synthesis
Calnexin-Calreticulin
chaperone i.e. HIV gp160 receptor binds to glucose! lectin properties released if protein loses glucose binds oligosaccharide with a terminal glucose to target it for degredation glucose usually trimmed when oligosccharide is added during N-linked glycosylation re-add glucose if misfolded so C-C binds
ERAD
ER associated degradation, to degrade misfolded or slowly folding proteins balance! if folds slowly –> more ERAD
ubiquitin
if protein is misfolded, retains chaperone, stays in ER translocated through membrane into cytoplasm (ATPase pulls it through) N-glycanase cleaves off glycoproteins ubiquitin ligase adds ubiquitin covalently to LYSINE (K) tags for degradation to proteasome (ubiquitin recycled)
del508 mutation
mutant CF gene poor folding and OFF (ERAD) is predominant way lacks channel so can’t regulate the ion concentration in the airways and can’t clear mucus
gain-of-function mutations
most misfolded proteins are loss-of-function some - lead to toxic accumulation of misfolded proteins, like Alzheimers, T2D –> toxic, ER stress –> trigger apoptosis
Synuclein
in fruit flies failure to fold is toxic - if overexpress, don’t fold, kill neurons if add chaperones –> mediate folding in the cytoplasm
ER stress
an imbalance btw capacity of the ER to process client proteins and load of proteins imposed on the organelle capacity: chaperones, oxidoreductases, glycosylation, protein degredation, lipids (membrane) demand: physio load, mutant proteins
ATF6, IRE1, PERK
tell when ER stress - when proteins not folding when activated –> turns on protein synthesis of what you need and inhibit what you don’t decreases load on ER but increase expression of genes that the ER needs rectifying response to ER stress
PERK
if making insulin - it’s all accumulating in the ER - not enough to make it and not enough capacity in ERAD! accumulate and cells die if no PERK - cells are destroyed! (ER response signal)
autophagy
alternate method to get rid of accumulated misfolding proteins in in ER ER–> under stress –> engulf part of cytoplasm with ER and contents –> merge with lysosome when not enough capacity and degrade by acid hydrolysis
COPII
ER to golgi smooth ER has exit sites with stable proteins made that turn into vesicles
COPI
golgi to ER
Sec23/24
inner coat proteins - binds to cytoplasmic tails of seected membrane proteins
Sec13/31
outercoat - able to deform membrane as binds and proteins old - area the COPII vesicles will form
ERGIC
clotting factors in vesicles - hemophilia if not secreted correctly
KDEL
COPI vesicles on things that need to cycle back from cis golgi to ER (chaperones have) These proteins are not free to diffuse into areas of COP II vesicles and if escape, brought back target sequence on peptides that need to stay in the ER, will be retrieved from golgi if there KDEL receptors have KKXX on receptor in COPI vesicle retrieves BIP from CGN
modification of core oligosaccharides in golgi apparatus
- oligosaccharide is added to Asn residue in the ER
- in the ER - glucosidase I and II removes 3 glucose, ER mannosidase removes 1 mannose, moves to golgi lumen
- in golgi - golgi mannosidase I removes 3 mannose - high mannose oligosaccharide!
- N-acetylglucosamine transferase I adds GlcNAc
- golgi mannosidase II removes 2 mannose
- add NANA (sialic acid) Gal, GlcNAc - very negatively charged! complex oligosaccaride
0-glycosylation
side chains built in golgi! can occur on lipids and proteins
on proteins: ser/thr - hydroxyl group
can add A and B group on proteins for antigens
enzymes in cisternae of proteins
CGN
sorting!
phosphorylation of oligosaccarides on lysosomal proteins
cis cisterna
removal of Man
medial cisterna
removal of Man
addition of GlcNAc
trans cisterna
addition of Gal
addition of NANA
TGN
sulfation of tyrosines and carbohdrates
sorting!
Furin
in TGN
catalyzes cleavage and activation of many proteins
removes sections to activate the proteins
i. e. HIVGP-160: only active when cleaved! Furin cleaves at certain basic AA
i. e. insuli receptor - cleaves it to activate it
transport of soluble lysosomal enzymes
TGN –> endosomes
protein from ER: add p-GlcNAc in CGN (phosphotransferase) –> cleave sugar and leave phosphate mannose –> uncovers M6P signal –> TGN –> bind to M6PR –> recruits clatharin coat –> transport vesicle –> fuse with early endosome –> dissassociate at low pH –> remove phosphate –> retromer coat to bring back
maturation of secretory vesicles
- proteins leave TGN
- acidic - aggregates
- membrane pinches off
- more acidic –> more aggregated –> remove membrane
- mature
PC
prohormone congertase
for all peptide hormones produced in this fashion
in immature secretory vesicles –> decrease pH for aggregating –> cleave proinsulin to insulin
cleavage of prohormone precursors in immature granules
how are vesicles targeted?
- microtubules! cytoskeleton can direct vesicles to their target membranes using microtubule based motor proteins that specifically bind to different vesicular carriers
- docking proteins control initial binding of vesicles to their target membranes
- SNARE proteins - specificity!
what cells traffic with microtubules?
- fibroblasts
- neurons
dyenin - goes in one direction, recruits different MT motors
Rab proteins
Rab-GTP is on vesicles
RAB-GTPase regulates docking! long protein on target, when there is membrane fusion, GTP is hydrolyzed.
each vesicle needs a specific Rab protein to know where it goes
GEF
guanine-nucleotide exchange factor
on donor compartment, exchanges GDP for GTP on rab for a new vesicle
VAMPS
V-Snares - only fuse w right t-snare
on vesicle
STX
T-snares - target snares! only fuse w right v-snare
recruit cargo
Mast cells
in connective tissues
if increase Ca2+ –> synaptic vesicles dock and fuse –> unregulated! asthma and allergies
neuron synapse
action potential - increase in ca 2+ -snare proteins fuse - secretory granules
Synaptotagmin
on vesicles - binds Ca2+ and regulates SNARE complex
Ca2+ binding in synaptic vesicles binds V-snares and prevents fusion - when Ca2+ releases V and helps the complex form
on vesicle!! inserted into membrane when bound to Ca2+, pulls plasma membrane up under the vesicle so there is easier fusion
Botox for migraines
relaxes muscles in head - inhibit synaptic transmission 0 cleaves snares so no vesicle fusion
phagocytosis
cell eating
pinocytosis
cells drinking - small particles, gulp large volume
clathrin triskeleton
3 clathrin heavy chains, 3 clathrin light chains
spontaneously form “cage” in cytosol
adaptor proteins
4 diff polypeptides - heterotrameric
bind clathrin and associate with membrane - bring clathrin to the membrane
membrane binding domain, binds to cargo (core)
clathrin binding domain (hinge)
accessory protein binding (appendage)
AP2
core plasma membrane adaptor for clathrin
rec specfic linear signal XXXL, YXX
mu2 subunit and alpha/sigma 2 subunits recognize
AP1
core plasma membrane adaptor for clathrin on TGN and endosomes
Dynamin
assembles into a ring around neck of forming bud - recruits other accessory proteins which destabilize lipid bilayer and vesicle pinches off
recruits GTPase - squeeze - provides E for the fusion of membranes and pinches
mutations in dynamin - deeply invaginated that can’t detach
sorting/early endosomes
pH about 6
ligand and receptor dissassociate
receptor in tubule, will be recycled
ligand will be degraded
pways of endosome/lysosome system
LDL
constituitive endocytosis!
LDL binds cholesterol, binds LDLR, endocytosed (clatharin)
sorting endosome - (low pH), LDL falls off of LDLR, lysosome, degrade LDL and recycle receptor
in lysosome - NPC1 and 2 - bind cholesterol and exit from digestive enorganelles
LDL - when cell needs cholesterol - put LDLR into PM
LDLR associates with clatharin coated pits
transferrin
constituitive endocytosis!
iron
nutrient uptake
temporally regulated
Fe3+ binds to transferrin - endocytosed (clathrin) –> endosome –> change conf and release Fe - high affinity for receptor and recycle back to surface
Apo-tranferrin (no iron) into liposome
NPC1 and 2
takes cholesterol out of lysosome
Familial hypercholeterolemia
increased LDL in bloodstream
devective LDL internalization
autosomal recessive hypercholesterolemia
defective adaptor protein - receptors are fine but adaptors are not
PCSK9
protein secreted by hepatocyte to regulate surface expression of LDLR
when endocytosed - degrade LDLR in the lysosome with LDL
can’t be recycled!
treatment: antibodies for PCSK9 - not there, can’t decrease LDLR -
EGF
ligand induced signal transduction
cytoplasmic downregulation of receptor tyrosine kinases - only when ligand binds to R
EGFR is regulated by ubiquitin and ESCRTs - cancer if not downreg
ubiquitin
ligases - tail added to R before it’s endocytosed to prevent it from entering the tubules - invaginating body pinches off and brings into lumen so isolated from cytosol
merge w lysosomal protease/lipase,
cleave
ESCRT
proteins that recognize ubiquitylated membrane proteins and sort into internal vesicles of multivesciular body
ESCRT -0,1,2,3, - bend endosomal membrane to form lumen - how downregulate membrane protens
GLUT4
Glucose transporter - major insulin regulator in muscle/adipose
stored in recycling endosomes and translocates in respose to insulin to increase glucose intake
T2D - insulin but no response!
Zipper mechanism
mechanism used by bacteria to induce phagocytosis by nonphagocytic host cells
bacteria expresses adhesion protein that binds w high affinity to a protein that usually binds to another cell (integrins, cadherins) i.e. adhesin bind to cadherin on host cell
form cell junction - move actin into cell etc, phagocytose
trigger mechanism
bacteria injects effector molecule into host that activates Rho-family GTPases –> actin polymerization –> ruffle etc. –> throw up large actin protrusions –> trap
anthrax
B subunit binds to receptor - cleaved - large subunit remains bound to R
7 B complexes form ring on target cell
Subunit A binds to the ring - endycytosed - in early –> late endosomes
decreased pH in endosome - ring conf change - pore in membrane - A enters cytosol
Ebola
ebola bnds - endocytosed –> early/late endosomes –> host cysteine proteases cleave GP and remove mucin and glycan cap - expose NPC1 binding domain
cleaved GP binds to NPC1 domain on lysosome compartment
triggers - conf change - viral membrane fusion and release in cytoplasm
LIMPS
highly glycosylated proteins on the lysosome - so the hydrolases won’t break down lysossome protein - sugar layer
lysosomal integral membrane proteins are highly glycosylated
hydrogen pump
ATP driven - maintains acidity with ATP
M6P
marker on all newly synthesized lysosomal hydrolases
N-linked oligosaccharide - put M6P on it!
GlcNAc phosphotransferase
in golgi
lysosomal hydrolase was was N-glycosolated in ER on Asp residue
adds UDO-GlcNAc to lysosomal hydrolase in the golgi
recognizes signal patch of lysosomal hydrolase and attach GlcNAc-P to mannose in oligosaccaride –> removes GlcNAc so only P remaining (uncovering of M-6-P signal)
M6PR
after M-6-P signal uncovered - binds to M6PR in trans golgi network - clatharin coated vesicle - merge w endosomes and dissasociate from R bc low pH
phosphotase removes phosphate (marker)
M6PR go back to TGN in retromer vesicle
MPR300
major transmembrane glycoprotein w 2 M6P bind sites and bind site for IGFII at repeat 11
bring hydrolases with M6P
LIMP-2
lysosomal integral membrane protein - heavily N-glycoslyated
trafficking in M6P in independent manor
1 way - doesn’t return
binds beta-GC from TGN to lysosome
Tay Sachs
mutations in genes that encode catabolic enzymes involved in the degredation of macromolecules
deficiency of Hex A activity caused by HexA gene encoding alpha subunit
Hex B activity is normal or increased
MPR46
minor MP6PR - only 1 bind site, dimer for increased affinity
non covalent dimers
bring hydrolases with M6P
sortillin
transport receptor for NP, Type I transmembrane, bring things to lysosomes
I-cell disease
defects in post-translational processing of lysosomal enzymes
errors in enzyme traficking/targeting
NPC disease
defective function of non-enzymatic lysosomal transmembrane and soluble proteins (transport of cholesterol out of lysosomes)
GM2 ganglioside
ganglioside with sialic acid, storage material in the brain
HexA
to break down Gm2 - cleaves it in the lysosome
alpha and beta subunits with Gm2 activator protein
Need Hex A and Hex B to break down Gm2 ganglioside
glycolipid bind site - lifts and extracts GM2 (ganglioside) from membrane - forms complex and hydrolyzes
beta-hexosaminidase system
need 2 polypeptides - alpha and beta and GM2 activator protein
Sandhoff
deficiency of Hex A and Hex B activities by mutations in HexB gene that encode beta subunit common to HexA and HexB
AB variant
mutations of MG2A gene encoding Gm2 activator protein - Hex A and Hex B ativities are normal but no hydrolysis of Gm2
ML II/III
lysosomal disorder
deficiencey of GalNac-PT - enzyme in golgi that gives M6P marker to hydrolases
hydrolases don’t reach destination - undigested material in lysosomes
MSD
defect in post translational modification
genetic deficiency of FGE that oxidizes suflatases and makes them active
no sulfatases
NPCI
defect - loss of protein - can’t redistribute LDL-derived cholesterol from lysosome to ER and PM
metabolic cross-correction
addition of M6P - transport to lysosome
recaptured by same/neighboring via cell surface M6PR –> endocytosis –> lysosome
take up exogenous enzykme –> deliver to lysosome –> degrade stored substrate and restore homeostasis
infuze enzyme outside body and infuse through stem therapy, gene therapy, enzyme
NOT every cell needs to be corrected - small amt makes big diff
substrate reduction therapy
lysosomes - drug that inhibits first step in glycosphingolipid biosynthesis, decrease rate of making to offset issues with breakdown
possible to cross blood brain barrier
pharmacological chaperone therapy
incrase folding/prevent premature degredation of defective lysosome enzyme
correct folding and decrese degredation in ER so gets to lysosome
even tiny percent increase is ok!
autophagy
self eating - ubiquitous proess in eukaryotic cells that results in breakdown of cytoplasm within lysosome in response to stress or to change
engulf organelles and merge w lysosome
macroautophagy
most common form of autophagy
phagophore/limiting membrane –> autophagosome –> autolysosome
chaperone mediated autophagy
Hsc70 binds to substrate protein in cytosol and thread through membrane of the lysosome
microautophagy
trapped in lysosome by invagination - usually non selected
lysosomal exocytosis
Ca2+ enters cell –> lysosomes triggered to excrete contents and repair PM
CLEAR gene network
lysosomal proteins - coexpressed and regulated by TFEB
regulates lysosomal biogenesis and function
TFEB
transcriptonal factor that promotes lysosomal biogenesis
mTORC1 - controls cell growth in response to nutrients and growth factor
if adequate lysosomal function: mTOR (on lysosome) phosphorylates TFEB, triggers the binding of 14-3-3 protein to TFEB - keep TFEB in the cytoplasm –> no translation!
if inhibition of lysosomal function: decrease mtor - dephosphorylation of TFEB, no binding of 14-3-3, TFEB can ener nucleus and stimulate lysosomal genes
i.e. if not enough AA - TFEB works to turn on genes, degrade more proteins and make more AA
LYNUS
lysosomal nutrient sensing - on surface of lysosome
regulate proteins assosicated with mTOR
LYNS - includes ATPase and all involved with mTOR
if enough nutrients: TFEB binds w LYNUS - sense lysosomal nutrient level and phosphorylates TFEB to sequester it
if starvation - mTOR is released from LYNUS, inactive! can’t phosphorylate - TFEB enters nucleus and turns on more genes (including itself! autoregulatory loop for more degredation)
