3 types of coated vesicles?
Where do they transport materials?
1. Clathrin-coated: transport material from the plasma membrane and between endosomal and Golgi compartments
2. COPI-coated: transport material from the Golgi to the ER in retrograde direction
3. COPII-coated: transport material from the ER to the Golgi i
Major protein in clathrin coated vesicles?
Structure of the clathrin coat?
clathrin
each clathrin subunit consists of 3 large and 3 small polypeptide chains --> forms triskelion
triskelions assemble into a basketlike framework to form coated pits on the cytosolic surface of membranes
Adaptor proteins?
Function?
found in clathrin coated vesicles
-they bind the clathrin coat to the membrane
-they attract specific cargo receptors and the soluble proteins that interact with them
(each type of adaptor protein is specific for a different set of cargo receptors)
Steps of clathrin coated assembly:
1. Coat assembly: assembly of the coat introduces curvature into the membrane which leads to the formation of a coated bud
2. Cargo selection and recruitment: adaptor proteins bind both clathrin triskelions and membrane bound cargo receptors
3. Vesicle fo
AP2 adaptor protein example:
1. AP2 interacts with phosphoinositide PI(4,5)P2 in the cytosolic leaflet of the plasma membrane
2. AP2 rearranges so that its binding sites for cargo receptors are exposed
3. The AP2 complex binds to the cytosolic tails of cargo receptors that display sp
How do PIs (phosphatidylinositols) become PIPs (phosphatidylinositol phosphates)?
PIP kinases?
PIP phosphatases?
How do they contribute to specialized membrane domains?
How can they control protein binding?
they undergo rapid cycles of phosphorylation and dephosphorylation at the 3', 4' and 5' positions of their inositol sugar head groups
PIP kinase: phosphorylates PIs
PIP phosphatase: dephosphorylates PIs
distribution of PIs, PIPs, PIP kinases and PIP phosp
BAR domains?
How do they induce membrane bending?
Positive feedback in BAR domains?
crescent shaped membrane bending proteins that bind to and impose their shape on the underlying membrane via electrostatic interactions with the lipid head groups
they form coiled coils that have a positively charged inner surface which interacts with the
Dynamin?
What two domains do they contain?
What is required for their function?
Which type of dynamin is expressed in all tissues of the body?
soluble cytoplasmic protein that pinches off clathrin-coated vesicles
contains a PI(4,5)P2 binding domain that tethers the protein to the membrane
contains a GTPase domain that regulates the rate at which vesicles pinch from the membrane
GTP hydrolysis
dy
How does clathrin coat disassembly occur?
1. PIP phosphatase that is co-packaged into clathrin-coated vesicles depletes PI(4,5)P2 from the membrane which weakens the binding of the adaptor proteins
2. hsp70 chaperone functions as an uncoating ATPase which uses ATP to peel off the clathrin coat
What controls clathrin coat assembly?
1. Local production of PIPs
2. Coat recruitment GTPases
Coat recruitment GTPase?
Function?
What two proteins regulate the coat recruitment GTPases?
GTP binding protein
active with GTP bound, inactive with GDP bound
they control the assembly of clathrin coats on endosomes and the COPI and COPII coats on the Golgi and ER
1. guanine nucleotide exchange factors (GEF) activates the proteins (GDP --> GTP)
2 examples of coat recruitment GTPases?
Where are they found in high concentration?
ARF protens: assemble both COPI and clathrin coats at Golgi membranes
Sar1proteins: assemble COPII coats at the ER membrane
found in the cytosol in an inactive, GDP bound state
Steps of the formation of a COPII coated vesicle
1. Inactive, soluble Sar1 GDP binds to a Sar1 GEF in the ER membrane
2. Sar1 releases its GDP and binds GTP
3. Conformational change occurs and an amphiphilic helix is exposed and inserts into the cytoplasmic leaflet of the ER membrane
4. GTP bound Sar1 b
How do coat recruitment GTPases affect coat disassembly?
the hydrolysis of bound GTP to GDP causes the GTPase to change its conformation so that its hydrophobic tail pops out of the membrane and causes the vesicle's coat to disassemble
Rab proteins? Function?
Where are they found?
Inactive when? Where are they?
Active when? Where are they?
small GTPases in the cytoplasm that can bind to a particular membrane and help with specificity by tethering to the correct vesicles
inactive when GDP is bound and they are bound to Rab-GDP dissociation inhibitor (GDI)
they are soluble in the cytosol
acti
Steps of tethering of a transport vesicle to a target membrane
1. Rab effector proteins interact with Rab-GTPs located on the target membrane, vesicle membrane or both
2. SNARE proteins on the two membranes dock the vesicle to the target membrane and catalyze the fusion of the two lipid bilayers
3. Rab-GAP causes the
Rab5 function?
assembles on endosomes and mediates the capture of endocytic vesicles arriving from the plasma membrane
Steps of the formation of a Rab5 domain on the endosome membrane
1. Rab5-GEF on the endosome membrane binds to a Rab5 protein and causes it to exchange GDP for GTP
2. GDI is lost and GTP binding alters the conformation of the Rab protein, exposing an amphiphilic helix and a covalently attached lipid group which anchors
What is a Rab cascade?
Example of when this occurs?
the recruitment of sequentially acting Rab proteins, allows Rab domains to be disassembled and replaced by a different Rab domain
endosomal maturation (Rab5 is converted into Rab7)
Different types of SNARE proteins and their structure?
What happens when they interact?
What complex do they form?
v-SNARES: found on vesicle membranes
single polypeptide chain
t-SNARES: found on target membranes
composed of 3 proteins
when they interact, their helical domains wrap around the other and form a stable 4-helix bundle
trans-SNARE complex (locks the two me
How does v-SNARE and t-SNARE pairing ensure vesicles fuse with the correct membrane?
Where does the energy come from to catalyze membrane fusion?
it is highly specific
trans-SNARE complex uses the energy that is freed when the helices wrap around each other and water is squeezed out
What protein is involved in SNARE disassembly?
How does it function?
What other proteins aid in this process?
NSF protein: cycles between the membranes and cytosol and catalyzes disassembly process
uses energy from ATP hydrolysis to unravel the interactions between the SNARE proteins
SNAP accessory proteins
Ways proteins can enter transport vesicles and exit the ER
1. soluble proteins contain exit signals which bind to cargo receptors in the ER membrane
2. membrane proteins contain exit signals that adaptor proteins recognize
3. enter by bulk flow
Homotypic fusion?
Heterotypic fusion?
fusion of membranes from the same compartment
fusion of a membrane from one compartment to one on a different compartment
Vesicular tubular clusters?
How do clusters move?
the structures formed when ER-derived vesicles fuse with one another
consist of a compartment that is distinct from the ER
they move along microtubules
Retrieval pathway?
What must the ER resident proteins contain?
Steps of the retrieval pathway
Explain how KDEL receptors function
How can they have a change in affinity?
returns escaped ER resident proteins back to the ER
KDEL sequence at the C-terminus
KKXX sequence at the C-terminus
1. Soluble ER resident proteins bind to the KDEL receptor via their KDEL sequence
2. Proteins are packaged into COPI coated vesicles
KDEL r
What occurs in the different cisternae of the Golgi?
Where is glycosylation complete?
in the TGN
Two classes of N linked oligosaccharides that are attached to glycoproteins
1. Complex oligosaccharides: when the original N-linked oligosaccharide added in the ER is trimmed and further sugars are added
2. High-mannose oligosaccharides: sugars are only trimmed, no new sugars are added in the Golgi
Steps of oligosaccharide processing in the ER and the Golgi
1. In the ER, 3 glucoses are removed by glucosidases
2. In the ER, a specific mannose is removed by a mannosidase
3. Golgi mannosidase removes 3 more mannoses
4. N-acetylglucosamine transferase I adds an N-acetylglucosamine
5. Mannosidase II removes two a
O-linked glycosylation?
Enzyme that catalyzes this?
O-linked glycosylation occurs on _______ in the Golgi apparatus
addition of sugars added to the OH groups on serines or threonines
glycosyl transferase enzyme
mucins and proteoglycans
N-linked glycosylation?
O-linked glycosylation?
starts in the ER with a transfer of 14 sugar units from dolichol to Asn
occurs in all eukaryotes
sugars are added one by one by individual enzymes after proteins have left the ER
Purpose of glycosylation?
1. Promotes protein folding: makes them more soluble and prevents aggregation
2. Helps the protein bind to chaperones and lectins
3. Makes a protein more resistant to digestion by proteolytic enzymes
4. Mucus coat of lung and intestinal cells
Explain cisternal maturation model
Explain vesicle transport model
views the Golgi cisternae as dynamic structures that mature from early to late by acquiring then losing specific Golgi resident proteins
new cis cisternae continually form as vesicular tubular clusters arrive from the ER and then mature to become medial c
Lysosomes?
Where are they found?
What types of enzymes are found there?
pH of lysosome?
membrane enclosed organelles filled with soluble hydrolytic enzymes that digest macromolecules
found in all eukaryotic cells
acid hydrolases: enzymes that work best at acidic pH
4.5-5.0
Vacuolar H+ ATPase?
Where is it found?
uses the energy of ATP hydrolysis to pump H+ into the lysosome in order to maintain its acidic pH (V-type ATPase)
Explain lysosome formation (kiss and run concept)
1. Late endosomes fuse with preexisting lysosomes or preexisting endolysosomes
2. Endolysosomes are formed and they fuse with one another
3. Endolysosomes eventually mature into lysosomes as hydrolases to complete digestion of their contents
4 different pathways that deliver materials to lysosomes:
1. Endocytosis: macromolecules taken up from extracellular fluid
2. Phagocytosis: found in phagocytic cells, engulfment of large particles and microorganisms to form phagosomes
3. Macropinocytosis: nonspecific uptake of fluids, membrane and particles atta
Process of autophagy
cytoplasmic cargo becomes surrounded by a double membrane that assembles by the fusion of small vesicles to form an autophagosome that is fused with a lysosome
involves the ATG9 transmembrane protein and SNARE proteins
Nonselective autophagy?
Selective autophagy?
And when do they occur?
bulk portion of cytoplasm is captured in autophagosomes
occurs in starvation conditions
specific cargo is packaged into autophagosomes that tend to contain little cytosol
occurs when worn out organelles need to be degraded and to destroy invading microbes
Explain the pathway that delivers lysosomal enzymes from the TGN to lysosomes
1. Enzymes in the cis and trans Golgi network add M6P groups to lysosomal enzymes
2. Transmembrane M6P receptors bind to the M6P groups
3. M6P-tagged enzyme segregate from all other types of proteins in the TGN
4. Clathrin coated vesicles bud off from the
Retromer coated vesicles?
used to deliver M6P receptors in the endosome back to the TGN
Explain recognition of a lysosomal hydrolase
lysosomal hydrolases contain a signal patch that is recognized by a GlcNAc phosphotransferase
1. GlcNAc phosphotransferase's catalytic site binds both high mannose N-linked oligosaccharides and UDP-GlcNAc
2. GlcNAc phosphate is attached to mannose
3. GlcN
Process of endocytosis?
2 types of endocytosis? Where are they found?
material to be ingested is enclosed by a small portion of the plasma membrane
membrane invaginates and pinches off to form a endocytic vesicle containing the substance
1. Pinocytosis: when cells constantly form endocytic vesicles filled with small molecul
Explain the steps of endosome maturation
1. Endocytic vesicles fuse near the cell periphery with an early endosome
2. Tubular portions of the early endosome bud off vesicles that recycle endocytosed cargo back to the plasma membrane either directly or via recycling endosomes
3. Early endosome is
Multivesicular bodies?
Function?
patches of the maturing endosome membrane that invaginate into the endosome lumen and pinch off to form vesicles
they carry endocytosed membrane proteins that are to be degraded
they also carry the soluble content of early endosomes destined for late endo
How are proteins sorted into intralumenal vesicles in the endosome?
1. Ubiquitin tags help guide the proteins into clathrin coated vesicles in the plasma membrane
2. Ubiquitin tags are recognized again in the endosomal membrane by ESCRT protein complexes
3. Ubiquitylated cargo proteins are passed from one ESCRT complex to
Pinocytosis?
What types of coated vesicles form? From where do they form?
How does the membrane surface area remain the same?
when cells ingest portions of their plasma membrane in small pinocytic vesicles
Clathrin coated vesicles
they form from clathrin coated pits
the same amount of membrane being removed by endocytosis is being added to the cell surface by exocytosis
Macropinocytosis?
Where can it occur?
When does it occur?
Steps of the process
clathrin-independent endocytic mechanism
occurs in all animal cells
does not operate constantly, it is only induced by a response
1. Cell signaling event leads to change in actin dynamics which triggers formation of a ruffle
2. As the ruffles collapse bac
Receptor mediated endocytosis?
Which type of coated vesicles?
Example of receptor mediated endocytosis?
when macromolecules bind to complementary transmembrane receptor proteins which accumulate in coated pits and then enter the cell as receptor-macromolecule complexes
clathrin coated veiscles
cholesterol uptake, transferrin, EGF
What are low-density lipoproteins (LDLs)?
protein complex that carries cholesterol in the blood as cholesteryl esters
Explain the steps of cholesterol uptake
1. When a cell needs cholesterol, it makes transmembrane receptor proteins for LDL and inserts them into the plasma membrane
2. LDL receptors diffuse in the membrane until they associated with clathrin coated pits that are in the process of forming
3. AP2
Early endosome function?
What occurs there?
Describe their movement in the cell
main sorting station in the endocytic pathway
receptors dissociate from their ligands due to the acidic pH (LDL and LDL receptor)
they patrol the cytoplasm underlying the plasma membrane along microtubules and capture incoming vesicles
Transferrin?
Explain the Transferrin uptake pathway
transferrin: soluble protein that carries iron in the blood, binds to transferrin receptor on plasma membrane
1. Transferrin binds to transferrin receptors and is endocytosed and sent to the early endosome
2. Low pH of the early endosome causes transferri
Epidermal growth factor?
Explain its entry into the cell and what happens in the lysosome
What occurs once EGF first binds to its receptor?
small extracellular signal protein that stimulates epidermal and other cells to divide
EGF receptors accumulate in clathrin pits only after binding to EGF
both EGF and EGF receptors are degraded in the lysosome
EGF binding activates a signaling pathway wh
How does receptor down regulation occur?
receptors on the cell surface are covalently modified on their cytosolic side with ubiquitin
only one or a few ubiquitin molecules are added
addition of ubiquitin causes the receptors to gather in clathrin coated pits and become degraded in the lysosome
Transcytosis?
Where does it occur?
Example?
when endocytosed receptors end up in a different domain of the plasma membrane from where they came
occurs in polarized epithelial cells
how newborns obtain antibodies from their mother's milk
Possible fates for transmembrane receptorproteins that have been endocytosed?
1. Returned to the same plasma membrane domain from which they came
2. Sent to a recycling endosome to a different domain of the plasma membrane (transcytosis)
3. Sent to lysosomes for degradation
How do endosomes adjust the concentration of specific plasma membrane proteins?
Example?
glucose transporters responsible for uptake of glucose are stored in specialized recycling endosomes until the hormone insulin stimulates the cell to increase its rate of glucose uptake
when insulin is bound to the insulin receptor, transport vesicles rap
Phagocytosis?
Which cells does it occur in?
What do they do after they ingest material?
Residual bodies?
form of endocytosis which uses phagosomes to ingest large particles like microorganisms and dead cells
macrophages, neutrophils
they fuse with lysosomes and the material is degraded
indigestible materials that remain in the lysosomes that are secreted by
What types of molecules do phagocytosis receptors recognize?
How are living cells not phagocytosed?
1. Complement components: collaborate with antibodies in targeting microbes for destruction
2. Oligosaccharides on the surface of certain pathogens
3. Cells that have died by apoptosis: presence of phosphatidylserine on the outside of the cell
they have
Exocytosis?
2 functions?
transport from the TGN to the cell exterior
1. membrane proteins and lipids found in the vesicles provide new components for the cell's plasma membrane
2. soluble proteins inside the vesicles are secreted to the extracellular space
Formation of secretory vesicles?
1. Secretory proteins aggregate in the TGN
2. Proteins bud off in immature clathrin coated vesicles
3. Clathrin coat is removed when the vesicles become mature
4. Motor proteins propel the vesicles along microtubules to the edge of the cytoplasm
Constitutive transport vesicles?
Regulated transport vesicles?
Synaptic vesicles?
fuse with the plasma membrane once they arrive there
wait at the membrane until the cell receives a signal to secrete them and then they fuse
tiny specialized secretory vesicles they store neurotransmitter molecules in nerve cells
Trans-SNARE complex?
Structure?
What modulates the SNARE machinery at the synapse?
responsible for docking synaptic vesicles at the plasma membrane of nerve terminals
consists of 3 proteins:
v-SNARE synaptobrevin: transmembrane
t-SNARE syntaxin: transmembrane
t-SNARE SNAP25: peripheral
synaptotagmin and complexin
Explain the steps of exocytosis of synaptic vesicles
1. Synaptic vesicles dock at the membrane
2, SNARE bundle partially assembles which forms which draws the vesicle close to the membrane
3. SNARE bundle assembles further but the additional binding of complexin prevents fusion
4. When action potential arri
Explain how recycling of synaptic vesicles occurs
synaptic vesicles can form directly from endocytic vesicles
synaptic vesicles needed to be replenished very quickly
most synaptic vesicles are made by local recycling from the presynaptic plasma membrane in nerve terminals
instead of fusing with endosomes
What are polarized cells?
Two ways of sorting plasma membrane proteins in a polarized epithelial cell?
cells that have two or more molecularly and functionally distinct membrane domains
1. Direct pathway: proteins destined for different plasma membrane domains are sorted and packaged into different transport vesicles
2. Indirect pathway: protein is retriev