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biosynthetic secretory pathway delivers new proteins lipids, and cards to the PM or cell exterior



cells remove PM and deliver to internal compartments called endosomes. this is a form of protein and nutrient capture,


what defines an compartment

the cytosolic surface of compartment membranes carry molecular markers that provide specificity and serve as guidance for incoming vesicles


where do they get the markers?

by budding off from one compartment and transfer to another


how do cells segregate components into membrane domains

proteins are assembled into domains following the assembly of a special protein coat on the cytosolic surface


how do coated vesicles form

most vesicles form from specialized coated regions of membranes. the coat is a distinctive cage of proteins covering the cytosolic surface that directs vesicles whereto go. coat is shed before fusing


differentiate the inner from the outer layer of coated vesicles

the inner layer: concentrates membrane proteins in a patch and directs vesicle formation. it allows selection for molecules with in patch for transport
outer layer: molds and forms the vesicle, basket like lattice deforms membrane patch


What are the 3 types of vesicles?

clathrin coated: transport from golgi and from PM
COPI: transport from golgi to PH or ER (retrograde transport)
COPII: transport from ER to golgi


Clathrin Coated Vesicles

major component is clathrin, each subunit has 3 large and 3 small pp chains that form a triskelion
isolated triskelions spontaneously assemble into polyhedral cages in vitro


what are adaptor proteins

forms the second layer b/w clathrin and the membrane. they link the clathrin coat to the membrane and trap transmembrane proteins. this keeps transmembrane proteins together with the soluble lumen proteins


What is the role of Adaptins in vesicle trafficking

each adaptor protein is specific for a different set of cargo receptors and for different membranes use different adaptor proteins and thereby have different cargos


what are phosphoinositides (PIP)

a signalling molecule.
has important regulatory functions associated with their phosphorylation states are 3',4' or 5'positions of the inositol head group.
conversion b/w PI and various PIP is highly regulated and compartmentalized by PI/POP kinases and PIP phosphatase distinct to different organelles


How are PIPs regulated

distribution of PIP can define specialized membrane domains.
proteins involved in vesicular transport will bind with high specificity to the inositol head group and they can even distinguish b/w different phosphorylated stages


define coincidence receptor

several conditions must be met simultaneously


what is the role of AP 2

binds to specific PIP leading to phosphorylation and conformational change that exposes cargo receptor binding sites allowing for the cargo receptor to bind


Visualizing PIP regulation

GFP fusion with a domain the binds PI(3,4,5)P can be used to visualize PI(3)P kinase activity at the membrane


How do vesicles pitch off

proteins are recruited to the neck of the bud (including dyamin) which regulates the pinching off of the bud.pinching off - 2 non cytosolic leaflets of the membrane are brought together and fuse


vesicle uncoaring

once a vesicle has pinched off, it sheds its clathrin coat to fuse with the target membrane.
a PIP phosphatase is packaged in the vesicle and depletes PI(4,5) from the membrane weakening the adaptor proteins
hsp 70can also function as an uncoating atpase activated by aucilin


what is role of Coat recruitment GTPase

to control assembly of clatrin on endosomes and COPI/COPII on golgi and ER membranes


GTP binding proteins

are molecular switches. use GEF to activate by catalyzing GDP > GTP and GAP to hydrolysis GTP > GDP


coat recruitment monomeric GTPase

ARF proteins (for COPI and clathrin) and SAR 1 (COPII) normally found in inactive GDP form in cytosol


how are COPII vesicles formed on ER

SAR 1_GEF is embedded in the ER membrane ad binds to cytosolic SAR 1 to switch GDP for GTP
now SAR 1 is active - a conformation change exposes an amphiphilic helix which allows SAR 1-GTP to insert into into the cytoplasmic leaflet of ER, once embedded SAR 1 recruits coat proteins that initiate binding


Forming COPII vesicles

First layer: SAR 1-GTP binds to two COPII proteins SEC23 and 24, 24 has binding sites for cytoplasmic tails of cargo receptors
Second layer: two other COPII proteins SEC 13 and 31 form the outer shell


COPII disassembly

mechanism is unknown b/c SAR-GAP is not identified SAR 1 may have a hydrolzer action
more stable than clathrin


What is tubular transport

endosomes and TGN were seen in live imaging to continually send out long tubules coat proteins assemble on tubules and recruit specific cargo


how do vesicles know where to go?

markers on their surface as an identification of origin and what they cary, target surface has complementary receptors



proteins act to direct vesicles to specific sites on target membrane they have an important role in specificity, part of ras super family
a single RAB can bing to multiple RAB effectors



proteins mediate the fusion of the lipid bilayer


RAB effector

facilitate vesicular transport, membrane tethering, and fusion can be many different thing


how are rabs activated

by membrane bound RAB-GEF found on both vesicular and target membranes
once RAB-GTP is membrane bound it can bind to rab effectors


what happens to snares after fussion

doesnt always occur after snares intertwine some require an increase of Ca+
snare complex must be disassembled and reactivated with the help of ATP, NSF protein and NSF accessory proteins


Protein folding/ assembly for exit

proteins that are mis folded stay in the ER b/c chaperones such as BiP and calnexin mask the exit signal


Vesicular tubular clusters in the ER

transport vesicles that bud from the ER shed their coats and can fuse with other transport vesicles
must have matching t/v snares


imaging of VTC

ER-Golgi intermediates
must used FRAP with two different fluorophores tagged to the cargo molecule


Retrograde/ retrieval transport

proteins with KDEL signal sequence are sent from the GA back to the ER are carried in COPI coated vesicles


oligrosaachride mod in goldi

enzymatic reactions occur on the inner membrane and all golgi glycosides and glycosyl transferases are single pass transmembrane proteins in multi-enzyme complexes


complex n-linked oligro

original n-lined core oligrosaacharide is trimmed and further sugars are added
if enzyme is accessible than complex occur


high mannose n-linked

trimmed but no new sugars are added in golgi


what i so linked glycosolation and sulfation

sugars added to -OH groups of Ser and Thr
with N-GalNAc
mucins are heavily glycosolated by o-linked
proteoglycans are one or mare glycosaminogyan chains, sugars are sulfated


Why glycosolate

protein folding: Nlinked makes intermediates more soluble prevents aggregation, progression of protein folding can be monitored by modification of attached saacrides in the ER
protection: bulky, non-flexible, and charged sugar chains can protect the cell from other macromolecules, the membrane protein can be protected from extracellular proteases and acidic molecules
cell-cell recognition: proteins called lectins or selectins bind sugars and function in cell-cell adhesion specificity of cell receptors can be affected by glycoslation


Golgi transport model 1
Cisternal maturation model

golgi is dynamic, cisternae move. at cis face vesicles fuse to form new cis cisternae which that matures and moves to become medial than trans


support for model 1

observations of large molecules too large to fit in vesicles, moving progressively through golgi stack and retrograde transport by COPI vesicles explains distribution of golgi enzymes


Model 2
Vesicular transport (stable compartment model)

golgi is static enzymes are held in place and molecules pass through transport vesicles, retrograde flow returns escaped ER and golgi proteins


Directional flow in model 2

cargo that needs to move forward can only access forward-moving vesicles
all vesicles are COPI coated but different adaptor proteins may confer selectivity
random input and output at cis/trans


the golgi matrix

a network on the cytosolic side that interact to bring vesicles in, tethering cargo similar to nuclear pore fibril


How is the architecture of the golgi matrix maintained

depends on MT cytosekelton and cytoplasmic golgi matrix proteins which form a scaffolding b/w adjacent cisternae/ stacks
Golgins form a long filamentous tether to guild transport vesicles


how is golgi divided b/w daughter cells

matrix proteins are phosphorylated at the onset of mitosis, then golgi fragments and disperses to allow for division of two daughter cells reversal by phosphatase allows reassembly



contain acid hydrolases
for optimal activity must be activated by proteolytic cleavage and h 4.5-5


lysosmal membrane proteins are

highly glycosolated: to provide protection
include many transporters that carry products of digestion out to cytosol
have a vacuole H+ ATPase that pumps H+ into lumen



resemble lysosomes but are not golgi derived
distinguished by paracrystallin structures inside a sac


How are lysosomes formed

formed from endosomes
late endosomes fuse with preexisting lysosomes (explaining diversity) latesendosomes can contain material from the PM or newly synthesized acid hydrolyses


yeast lysosomes

very large vacuoles 30-90 percent of cell volume
functions are more diverse: homeostasis of cytosolic pH nutrient and waste storage


plant lysosomes/vacuoles

controls cell size, turgor pressure and pH
adjust to large changes in tonicity by changing osmotic pressure in vacuole and cytosol: breakdown and resynthesis of polymers;change transport of sugars, aa, and other metabolites


how to demonstrate presence of lysosome is years culture

use a substrate (fluorophoe and DIC) that could be degraded by phosphates which would deposit a stain in the lysosome, use light microscopy to see


how are substrates delivered to lysosome

new digestive enzymes are made in ER and transported through golgi, the lysosomal compartment is the convergence point for intercellular traffic. enzymes meet their substrates in 4 ways: endocytosis, autophagy, and phagocytosis


Substrate delivery: Endocytosis

endocytic vesicles are first dilivered to early endosome, which fuses with vesicles arriving from the golgi (loaded with lysosomal hydrolyses). some endocytosed material is recycled to the PM other is passed onto the late endosomes
as late endosomes acidify and mature into lysosomes endosomal membrane proteins are recycled back to the endosomal compartments TGN


Substrate delivery: autophagy

the proteasome degradation system is too small for some jobs, autophaosomes degrade old mitochondria and large protein aggregates
there are specific proteins that initiate the process of autophagy for other organelles like the ER



form from unknown orgin, engults portion of cytosol syrounds target and forms a double membrane structure; then fusion with the lysosome/ late endosome contents are digested and then inner membrane of the autophagosome breaks down


substrate delivery: phagocytosis

macrophages are professional phagocytes and are specialized for the engulfment of large particles and microorganisms the resulting phagosome is converted to a lysosome by fusion with existing lysosomes


how are acid hydrolases delivered to the lysosomal compartment?

lysosomal proteins are co-translationally translocated into ER lumen than transported through the golgi to the TGN transport vesicles bud off TGN and are targeted to early endosomes
recognition involves M6P which is added to n-linked sugars of soluble lysosomal hydrolases as they mass through cis-golgi lumen


M6P receptor

integral proteins in TGN membranes which bind to M6P on lumenal side + adaptor proteins on cytosolic side to assemble CCV. the receptor binds to M6P at pH 6.5 and releases at 6.0
acid phosphatase remove P from M6P destroying signal
signal in M6PR cytoplasmic tails direct forward (CCV) and reverse (retromer coated transport vesicles) directions
some M6P escapes and is secreted to ECF, M6PR return through receptor mediated endocytosis


retromer coated vesicles

assembles on endosomes for return of M6PR to TGN


what causes the retromer to assemble

the retromer only assembles when:
it can bind to the cytoplasmic tails of cargo receptors, interact directly with a curved phospholipid bilayer, can bind a specific phophroylated (PI(3)P serves as an endosomal marker signal)
thought to function as a coincidence detector upon binding will stabilize membrane curvature
small in comparison to triskelion


how can we acquire M6P

through two successive enzymatic reactions:
1. N-acetylglucosamie - 1- phosphated (GlucNAc-1-P) added to one or more specific mannose residues in cis-golgi
2. GlcNAc is cleaved leaving mannose residues phosphorylated in 6th position


Lysosome storage disorderes

result in an accumulation of undigested material in lysosomes.
nervous system affected - fatal
mendelian inheritance - autosomal or x-linked recessive
lysosomes swell when they arnt able to break them down.


I-cell disease

inclusion-cell disease, all hydrolytic enzymes are missing from lysosomes due to mutation in GlcNAc-phosphotransferase, therefore, all lysosomal hydrolases fail to receive M6P signal and are secreted


Lysosomes are not always the final digestive stage

undigested debris can be expelled through lysosomal secretion


how do melanocytes used lysosomes

cell type with lysosomes that can fuse with PM
melanin is produced and stored in melanosomes, after fusion with PM melanin is released into the ECS and is taken up by the basal layer keratinocytes
melanin protects chromosome of mitotically active basal cells agaisnt light induced damage


how do endosomes mature

internalized cargo is sorted after formation of endocyotic vesicles and fusion with early endosomes,, return to PM via recycling endosoes or degradation cis inclusion in late endosome


multivesicular bodies

a subset of endosomes that contain membrane-bound intralumenal vesicle
formed before maturation into late endosom



vis CCV
also occurs caeolae: present in PH of most cells, deeply invaginated flasks, originated from lipid rafts contain caveolin



clathrin independent all animal cells, degradative fuses with lysosomes
induced by cell surface receptors activation, changes in actin dynamics forms ruffels when ruffles collapse form large fluid filled endocytic vesicles


LDL receptor mediated endocytosis

specific macromolecules bind to transmembrane receptors accumulated in coated pits and taken into the cell via CCV


example of LDLR uptake

uptake of cholesterol from blood.
internalized LDL +LDLR are delivered to early endosome, reduced pH causes disassociation of ligand and receptor. LDLR is returned to PM LDL remains in late endosome than fuses with lysosome
increase of cholesterol in cell shuts off synthesis of new LDLR


General description of receptor mediated endocytosis

distinct receptors internalize via signals in cytoplasmic tails, bind to adaptor proteins in clthrin coat
some receptors accociate with forming pits regardless of ligand
all vesicles are delivered to early endosome but different receptors are processed differently


Receptor Recycling

only receptor is recycled, both are recycled, both are degraded `


when only the receptor is recycled

returned to PM
DLDL disassociated for LDLR in slightly acidic endosomes > LDL goes into lysosome but LDLR returns to PH via CCV that bid from tubular region of early endosomes
can be visualized with FRAP


Receptor and ligand are both recycled

return to PM
Tf is a soluble protein requited for transporting iron in blood TfR binds to Tf and cycles to endosome where acid pH induces release of iron but Tf and TfR remain bound until recycled to the neutral pH of the PM


degradation of both

EGFRs internalize only after binding to EGF, ubiquitin binding proteins ensure receptor downregulation which leads to decrease in concentration of EGFR at surface


prove that a receptor can be recycled in the same way as tranferrin

fuse protein of study to GFP, and transfect cells. > excite at the right wavelength to see fluorescence >tag transfeerin with another colour > f they follow the same pathway you should see a mix of the two colours


prove that something is a recycling endosome

use a protein that we know recycles like transferrin and see if it is glowing as the right colour ??


characteristics of early endosomes

visualize endosome with Em using tracer like peroxides, need e-dense produce. relatively small and patrol under PM along MT capturing incoming vesicles


maturation of Early endosomes

change shape and location. rabs, pips snares are all part of the molecular makeover changing the functional characteristics
v-type ATPase acidifies organelle, instralumenal vesicles sequester endocytosed receptors
delivery of lysosomal proteins for TGN, hydrolases are more active in late endosome b/c the are in their optimal pH


Maturation into Multivesicular bodies

patches of endosomal membrane invaginate into lumen and pinch off to form intralumenal vesicles, migrate along MT towards cell interior, shed tubules and vesicles



sorting into internal vesicles needs 1+ ubiquitin tags added to the cytosolic side of membrane proteins >
ESCRT recognize ubiquitin tags
ESCRT is also involved in transport of cholesterol and PIP is also involved in vesiculating


clathrin-mediated endocytosis in real life

prepare slices for EM, image both using light and EM
find clathrin colocalization with CCV
use Tf as a marker for recycling
use FRAP to visualize recycling of clathrin



transporting proteins from one side of the cell to the other crossing the cell.


Example of transcytosis

Ab transfer from amma's milk to baby's intestinal epithelium.
gut lumen is acidic, promoting the binding of Ab to AbR. is taken up via CCV that delivers the complex to early endosome.
transport vesivcles bud off early endosome (with AbR and Ab complex) and is directed to intermediate/recycling endosome. upon fusion with basal PM the return to neutral pH causes disassociation


additional function of an recycling endosomes

can be a storehouse for PM and specialized transmembrane proteins. eg. glucose transported in to cells can be up regulated by release of glucose transporters from recycling endosomes



large endocytic vesicles by phargocyted and is cell mediated. important source of nutrients for Protozoa, few multicellular organism cells do this for purposed other than nutrition
fuse with lysosomes for degradation
not constitutive



fusion of golgi derived vesicles with PM - release to ECS or PM via CCV


reasons for exocytosis

secretion: of proteoglycan and glycoproteins for formation of ECM
constitutive secretory pathways: all cells have ongoing exocytosis
Regulated secretory pathway: specialized cells control release of certain soluble proteins or other substances


Main protein sorting pathways from TGN

in a cell that does regulated secretion there are 3 ways of passing through golgi:
target to lysosome
packaged into secretory vesicles
immediate delivery to PM and release


Secretory vesicles

specialized cells using regulated pathways to secrete and concentrate products for storage


how to determine what is being secreted

used Ab: anti clathrin and anti-insulin and look for overlap, cross link antibodies with different flourophores, if there is over lap yellow will be seen, then use immunofluorescence microscopy


maturation of secretory vesicles

aggregates are too large to be bound by simple receptors, resembles phagocytic uptake. secretory vesicles.
membrane retrieval: membrane recyclingreturns golgi components to golgi and helps concentrate


how to get secretory vesicles ready for release

motor proteins pull secretory vesicles along MT which guild vesicles along the default constitutive exocytosis pathway. once at release site a signal is needed to allow fusion with PM


What are synaptic vesicles

nerve and endocrine cells contain regular secretory vesicles and specialized synaptic vesicles which are smaller and store NT. complexins freeze snares in metastable state until there is a rise in Ca+ > synaptotagmin displaces complexin and allows for fusion


Steps of excocytosis of vesicles

docking: synaptic vesicle docks with the PM via V and T snares
Priming I:partially assembled snare bundle
Priming II: complexin blocks snare bundle from full association
Fusion pore opening: increase of Ca+ causes conformational change disloging complexin and allows for snares to fully associate
Fusion complet: once pore is opened, snares and associated proteins disassociated and NT is released


how do synaptic vesicles form

formed via recycling PM in nerve terminals; components are first delivered to PM by constitutive pathway and retired by endocytosis. vesicles are immediately reloaded
specialized membrane proteins facilate NT uptake from cytosol


Situations where PM must grow

cytokinisis, phagocytosis, PM repair, and cellurlarization


exocytosis in polarized cells

segregation occurs at TGN , apical surface enriched in GSL and GPI anchored proteins.
lipid rafts will form in TGN and PM vis self association of GSL and cholesterol.
basolateral proteins have sorting signal in cytosolic tails


chemiosmotic coupling: ETC

part one: High energy e- from oxidation food/photon excitation are transferred through a seres of membrane proteins forming the ETC. each electron transfer releases a small amount of energy which is used to pump H+ across the membrane generating the electrochemical gradient


Chemiosmotic coupling: ATPase

H+ flow back down the gradient through ATP synthase


steps of mt ETC

1. e- move through protein complexes via prosthetic groups like metals or other carrier bound to proteins
2. NAD+ +2e- =NADH, produced in krebs/citric acid cycle
3. Carbs
input:fats and carbs
output:water and CO2


Chloroplast ETC

1. e- move along protein complexes via prosthetic
2. photosystems use chlorophyll to capture light.
3. reactions run in opposite directions: e- from water used to generate oxygen via NADPH


mitocondria characteristics

associated with MT for orientation and distribution
found where ATP demand is high
associated with the ER: ER physically extend tubules that wrap around the mitochondria and are involved in assisting them divide and facilitate extange of lipids


5 functional spaces of the mitochondria

matrix: metabolic enzymes, oxidation of food stuff
IMM: machinery for OXPHOS metabolite transporters, TIM, also generates cristal space
OMM: many porin, TOMS, pro-apoptotic proteins
IMS equivalent to cytosol (same pH and ion concentration)
Cristal space


what is the Cristal space

cristae are membrane discs that protrude deeply into matrix the crista membrane is continuous with I


other mitochondrial functions

provide carbon skeletons and reducing power to cytosol
citrate is transported to cytosol to generate C and NADPH
metabolite shuttle system to buffer redox potential in cytosol
heme and lipid biogenesis


what is redox potential

measure of e- affinity as delta G,
midpoint values
NADH/NAD - 320mV
UQH2/UQ 30mV
red cyt/ox cytx 230 mV
h2o/o2 820mV


electron carriers in the respiratory chain

cytochrome = heme + apocytochrome
FeS = detect by e- paramagnetic response
flavin and copper atoms prosthetic groups


what are the 3 H+ pumping complexes

complex 1 : passes electrons to CoQ
complex 3: accepts e- from CoQ and passes to cytochrome C
Complex 4: 2 Cu uses 2H+ and 1 O to make water


what experiment can you prepose to see in a yeast has a mt defect

grow on glucose and ethanol/non-fermentable C source. yeast can grow in absence of o2 or in presence of mt defect. will not grow on ethonol


respiratory chain supercomplexes

aggregate together in IMM facilitates the efficiency of e- transport in cristae membrane


what drive OXPHOS

favourable ATP/ADP ratio much be maintainedonly in presence of high [atp] hydrolysis of P bond occurs


what is F1F0 ATP ase

nonomachine that generates ATP
F0 creates hydrophilic pathway for H+
mechanical energy is converted into chemical bond energy


how is atp sythase also an atpase

molecular motor can work in reverse - use energy form ATP hydrolysis to pump H+ back


what is the role of Crisae in ATP syntesis

EM shows that ATP synthase on matrix side of crita forms long rows of dimers to induce and stabilize regions of membrane curvature.
ETC h+ pumps on either side of dimer cristae functions as H+ traps


H+ gradient coupled transport accrues IMM

couple flow of H+ to the transport of needed metabolites like pyruvate, PI, ADP, and ATP. 4atp out 3 adp in.


respiratory control

uncoupling agents disconnects the e- transfer from atp production to increase O2 uptake


H+ gradient produced bulk of cellular atp

motile bacteria also use a gradient to drive propulsion
favourable ratio atp:10 adp drives the synthesis (delta G is very negative)
can use S,C, N rather than O2 serious of e- carriers in Pm similar to IMM in eukaryotes


how do organellar genomes resemble bacterial DNA

cp DNA has similarities to prokaryotic DNA, ribosomes similar in structure and antibiotic sensitivity translation starts with n-formyl methionine
mtDNA has fewer similarities similar antibiotic sensitivity, translation starts with n-formyl methionine, polycistronic messages, tRNAs serving as punctuation b.w protein coding gene regions
transcription /translation/replication take place in organelle
5 genes shared amongst al mt genomes


mitochondrial biogenesis by fission

growth, replication and duplication of mt is independent of the cell cycle will replicate according to metabolic needs of the cell . mt DNA is found on the matrix side of the IMM


what are dual genomic contributions to OXPHOS

some subunits for the OXPHOS complexes are mtDNA encoded, most are nuclear encoded. complex 2 is the exception b/c non of the subunits are encoded in mt genome.
proteins that control mt DNA are nuclear encoded, but these proteins are not the same as those involved in the nuclear genome


how is oracular DNA inherited

non-mendelian, in yeast in biparental fashion in plans and animals uniparental - maternal


other functions of mtDNA

1. dense gene packaging - in humans no introns
2. relaxed codon usage
3. varient gentic code - UGA isnt stop
4. increased mutation rate


mt heteroplasty

not all mtDNA molecules in a mitochondria or in a cell are all the same
as a result we see threshold effects
sandome segregation of mtDNA during division leads to heteroplamy


threshold effects

mutants load doesnt elicit the sam phenotypic effect on different tissue


what causes mt diseases in humans

mtDNA- maternal inheritance
nDNA -autosomal recessive/dominant
OXPHOS machinery can be affected leading to:
primary PSPHOS disorder : largest subset of mt diseases
secondary OXPHOS dysfunction


what does expression of mutant mtDNA depend on

tissue type
mtDNA depletion or multiple mtDNA deletions are a cause of mutation in the nuclear gene encoding proteins (dominant)
OXPHOS - one or morse respiratory chain enzymes deficient (recessive)
symptoms: neurodegradation, liver failure, developmental delay