BI 256: 13/membrane trafficking

What happens to anything that leaves the cell?

vesicles fuse with plasma membrane to release contents outside, lumen topologically equivalent to outside of the cell but CYTOPLASM STAYS AS CYTOPLASM

What is a vesicle?

small membrane-enclosed compartment, containing cargo

can leave via exocytosis, fuse with a membrane via endocytosis

What is clathrin?

crucial protein involved in endocytosis, triskelion, 3 heavy & light polypeptide chains, forms clathrin-coated pits

What are clathrin-coated pits?

Specialized regions of the plasma membrane where clathrin assembles to facilitate the internalization of molecules through endocytosis, done in an aqueous environment, no need for membrane

need nothing but clathrin to make pits, nature of shape forms structure

What does Clathrin need to work?

nothing but it’s ability to form Clathrin pits, nature of the shape forms the structure, triskelions spontaneously assembly in a symmetrical basket structure, in vitro & without membrane

What state are vesicles in when released from the membrane?

transported naked to show signals for future receptors & processes, no energy to take PIP & Clathrin

What is the main function of clathrin?

invaginating membrane, forming vesicles to transport substances into substance without energy

What are adaptins?

• adaptor proteins forming an inner vesicle coat

• bind clathrin coat to membrane & start to recruit for pathway

• trap transmembrane proteins to capture soluble cargo molecules inside vesicle

• help bridge gap between clathrin and receptor so clathrin can do invagination

How is clathrin recruited?

recruited to plasma membrane for endocytosis by recognizing specific phosphorylation patterns/phosphoinositides (PiPs)

What are the steps for Clathrin-coat assembly?

1. coat assembly & cargo selection, PI(4,5)P2 binds to donor membrane & cargo receptors, Calthrin binds

2. bud formation, Clathrin & adaptins bind to Clathrin

3. vesicle formation/pinching off using Dynamin

4. uncoating, no energy to take all off

What does Clathrin work with?

relies on a series of adapter proteins & membrane-bending proteins to make transport vesicles, bind Clathrin to cargo receptors, determine which substances are internalized

Does Clathrin interact with PIPs?

Yes, has an affinity for it that brings Clathrin to plasma membrane

What does clathrin coat assembly induce?

curvature, forces membrane to do invagination/pull into cell, rigid basket of triskelion forces membrane to bulge inward, pull into cell

What happens once the vesicle forms?

downstream proteins (dynamin) involved in pinching off the vesicle for transport

What is dynamin?

• enzyme, pinches off vesicles in endocytosis

• hydrolyzes GTP to facilitate pinching off

• has a PI(4,5)P2 binding domain

How does dynamin work?

• wraps around stem of vesicle left by Clathrin, forces 2 inside leaflets of vesicle membrane to fuse, expels water out to fuse

• regulates rate of pinching off with GTPase domain, finishes off the budding

When & why is the clathrin coat stripped?

after vesicle budding, to allow fusion with target membranes and release of cargo, receptors on surface are needed for further use

coat is just clathrin & adaptins, shedding exposes markers to tell where to go

What helps to strip off the clathrin coat?

phosphatases, dephosphorylate PIPs to weaken clathrin & adaptor protein binding

chaperones, use ATP to fully take off the coat

• dynamic is a bully, stripping off clathrins is energy efficient

How is Clathrin uncoated?

1. PIP phosphatase inside the vesicle depletes/dephosphorylates PI(4,5)P2 from membrane, weakens adaptor protein binding to PIPs

2. Hsp70 chaperones in cytosol act as ATPase, force clathrin coat off

happens at plasma membrane

What would happen in the Clathrin uncoating process without a GTPase?

can’t pinch off, formed veiscles but are stuck from no coat removal

What is the cargo selection process?

• specific cargo selection to avoid uptake of random materials, ensure cellular efficiency

• cargo receptors bind to transmembrane proteins/soluble ligands outside the cell

• Sec proteins help to bridge cargo receptors to clathrin

What does COPII vesicle assembly need?

Sar1 coat recruitment GTPase

• inactive Sar1-GDP in cytosol converted to Sar1-GTP using a GEF in ER membrane

• GDP to GDP conversion exposes an amphiphilic helix (GTP bound state)/tail, tailembeds Sar1 in ER membrane & is active

What does Sar1-GTP recruit?

adaptor proteins, binds to Sec23/24, 24 binds to cargo receptors & acts as an adaptin, 23 to Sar1-GTP, become inner coat, start invaginating membrane

What do Sec 23/24 recruit?

outer coat proteins, Sec13/31, GTPase will clip off once outer and inner layers formed

What is the COPII pathway?

anterograde, deals with the transport of materials from the ER to the Golgi apparatus

• GTPase SAR1 activates by binding to GTP at ER membrane

• SAR1 activates, recruits Sec23 & 24 to help vesicle formation & cargo selection from ER

What is the difference between the clathrin & COPII pathways?

clathrin can form structures independently, COPII needs multiple proteins for vesicle formation

COPII vesicle release is less defined, SAR1 acts as a molecular timer guiding disassembly

What is the uncoating process of COPII?

COPII coats stimulate GTPase activity of Sar1, leading to hydrolysis and coat disassembly, Sar1-GDP is inactive & cytosolic again

done to know where to transport

What molecular timer mechanism goes COPII use?

amphiphilic helix/tail in is timer, pulling out of the membrane has the coat fall off, tells Sar1 to stay in GTP state enough for vesicle formation

What keeps Sar1 from creating vesicles in places other than the ER?

localization of GEF leads to a cascade of events that promotes Sar1 activation specifically at the ER membrane, preventing random vesicle formation elsewhere.

What is the difference between Sar1-GTP & dynamin?

Dynamin doesnt matter how fast or slow, wants to be fast with hydrolyzing, Sar1 wants to be slower to get coat formed then do hydrolysis

T or F: vesicles are always round.

False, shape of vesicle can be tubular to accommodate larger cargo

ex- procollagen

What is the COPI pathway?

retrieves escaped proteins from the Golgi back to the ER, same as COPII but ARF1-GDP & GTP, instead of SAR1, other components assumed but unknown

What happens if you bypass the GEF in COPI & COPII?

Bypassing the GEF in COPI and COPII pathways prevents the activation of Sar1 and ARF1, leading to impaired coat assembly and vesicle formation, no localization and can go anywhere

What is KDEL/ER retention?

accidentally let some KDEL proteins out of ER to Golgi, retrieval path to bring back (COPI)

What must all vesicles be?

selective and targeted, not random, specific for transmembrane & soluble cargos

What determines the final destination of vesicles?

markers on surfaces of vesicles, specifically selection signals that guide their transport for transmembrane cargo or soluble cargo

What is PI?

phosphatidylinositol, a phospholipid that plays a key role in cell signaling and membrane trafficking

What is the 1st level of specificity for vesicle trafficking?

phospholipids across the cell

What are PIPs?

phosphatidylinositol phosphates, pattern of phosphorylation tells where they are in the cell

Why are PIPs used?

phosphorylation pattern on inositol ring determines where in the cell PIP is found, tells the cell what processes are being done & which proteins are included, and helps in the recognition and fusion of vesicles with their target membranes.

What part of PIPs can be phosphorylated?

ring structure can be phosphorylated by any of C, done by different kinases ar different parts of cell will determine which C get phosphorylated, coat proteins then bind PIPs to target vesicles

Which PIP is mainly used in vesicle trafficking?

PI(4,5)P2, used in the membrane to recruit specific proteins involved in vesicle formation and fusion, membrane composition directs vesicle trafficking

Why are there different coat proteins for different compartments?

Different coat proteins ensure that vesicles can selectively transport cargo to specific destinations within the cell, as each compartment has distinct protein and lipid compositions.

What is the COP1 Pathway?

retrograde transport, involved in retrieval pathways, moves things from the Golgi to the ER, coat protein complex makes vesicles for organelle ID & function

What does the COP1 pathway ensure?

proteins with ER retention signals are returned, maintain cellular homeostasis, prevent essential components depleting

What is the COP2 pathway?

anterograde transport, transports folded & modified proteins from ER to the Golgi, facilitates processing & sorting before final destinations

uses coat protein complex to form vesicles to help deliver proteins & lipids

• Properly folded proteins exit the ER via COP II vesicles. At the Golgi, COP I-mediated retrieval pathways correct mislocalized proteins, emphasizing the critical importance of fidelity during protein localization processes.

What does SAR use to work?

GTP & GDP exchange, GTP activated helps vesicle formation & cargo selection at membrane compartments, GTP bound state of proteins triggers assembly of coat proteins around budding vesicles

What is the importance of hydrolysis?

converting GTP to GDP is crucial for vesicle detachment & membrane fusion

What would impairing hydrolysis cause?

improper vesicle targeting & cargo mis delivery, causes cellular dysfunction

What is the secretory pathway?

exocytic, ER to Golgi to vesicles outside of the cell, vesicles start in ER, and go around and back

What is the retrieval pathway?

COPI, transports escaped or mislocalized proteins from the Golgi back to the ER, preventing accumulation of non-functional proteins and ensuring a well-organized cellular environment, KDEL/ER retention, KDEL slipped out of ER to Golgi, bringing back to ER

What is the delivery pathway?

COPII, proteins are accurately delivered to their designated target locations within the cell, which is essential for intracellular communication and metabolic processes

What are RAB proteins?

GTPases on membrane surfaces, guide vesicles to membranes & help with directionality & cargo localization, why we uncoat, complementary pairs with vesicles, >60 known in different organelles, don’t know how they’re incorporated into correct membranes

Where are inactive Rabs found?

In the cytosol, bound to GDP, and GDI, GDP dissociation inhibitor to keep inactivated

How are Rabs activated?

activated by guanine nucleotide exchange factors (GEFs) on vesicle & target membranes making GDP to GTP & releasing GDI, active Rab-GTP binds to membranes to bring vesicle to dock

How do Rab proteins guide vesicles?

target vesicles, tether to Rab effector using its specificity for directionality in transport

What is the Rab effector?

grabs vesicle & Rab-GTP and brings closer to snares to work, can be motor proteins & tethering proteins

• motor → move vesicles along cytoskeleton

• tethering → linking 2 membranes

What is the advantage of using RAB?

RAB proteins enhance the specificity and efficiency of vesicle trafficking by ensuring that vesicles fuse only with the correct target membranes, facilitating accurate delivery of cargo

What mediates membrane fusion?

SNAREs, v-SNARE on vesicle membrane & t-SNARE on target membrane, fuse vesicle with target compartment

What are SNAREs?

SNARE proteins facilitate the fusion of vesicles with target membranes, acting similarly to twist ties that secure bags, which helps in expelling water between adjacent membranes and promotes effective fusion, release cargo into correct area/compartment

How do SNAREs mediate membrane fusion?

1. Rab effector brings vesicle to t-SNARE complex on target membrane, GTP converted to GDP & left in cytosol with GDI

2. v & t-SNAREs twist so tightly fusion happens, energetically favorable from affinity of proteins for each other

3. This process triggers the formation of a stable SNARE complex, allowing the membranes to merge and release the vesicle's contents.

4. Rab is released & recycled

What action of SNAREs needs energy?

disassembly of SNAREs, disrupting proteins’ affinities for each other/helical bundle, needed so SNAREs don’t end up in target compartment, uses NSF an ATPase to pull apart then recycle each

How many SNAREs are there in animal cells?

at least 35

How do SNARE's maintain topological equivalence?

maintains the internal configuration of cargo throughout transport, preventing exposure of cargo to the cytosolic environment, which can lead to degradation or miscommunication within the cell.

What relevance does membrane fusion have in reproduction?

membrane fusion processes that occur during fertilization are reminiscent of those involved in vesicular transport and are crucial for successful reproductive functions, such as sperm-egg fusion

How do viruses use membrane fusion?

envelope viruses fuse with our cell surfaces, use own SNAREs (none outside0

How do COPII vesicles exit the ER?

use ER exit sites (smooth & w/o ribosome), leave with exit signals on cargo receptor, membrane cargo interact with coat (13/31 & 23/24), soluble cargo interacts indirectly

Do COPII vesicles always use exit signals?

try to, sometimes can leak out without it accidentally

What is the role of microtubules in vesicle transport?

all vesicles are actively transported along microtubules via motor proteins (dynein and kinesin), which helps in preventing congestion within the cell while ensuring efficient distribution of vesicular cargo

Can ER vesicles fuse en route to the Golgi?

Yes, use less energy, being moved along microtubule highway with motor proteins, ER vesices form VTCs

What are VTCS?

vesicular tubular clusters, bud off own transport vesicles, return escaped proteins & proteins needed only for budding back to ER (retrieval)

made & start to activate retrieval transport for checking anything not in cluster/leaked out

strip off coat, fuse vesicles to make VTCs using snares, 1 cluster relies on 1 motor protein to move

How are VTCs formed?

vesicles can fuse together, reduces the energy required for transport. This clustering allows for the pooling of resources and more efficient handling of multiple vesicles at once.

How does the KDEL signal sequence work?

used to identify soluble ER resident proteins that should remain in the ER. KDEL receptors play an essential role in retrieving mislocated proteins from the Golgi or VTCs and returning them to the ER

How are KDEL signals used?

usually for staying in cell, can help in retrieve ER proteins in COPI vesicles, leaked out in accidental transport vesicles, bound by KDEL receptors & sent back to ER with COPI vesicles from VTC

What environmental influences work on KDEL sequences?

slight change in acidity as proteins transition from the ER to the Golgi affects the binding affinity of KDEL receptors for their cargo

Why does pH impact KDEL receptors?

pH sensitivity is crucial for the selective retrieval of proteins based on their localization

Why are RABs & SNAREs used?

vesicle traffic control, precise targeting and fusion of vesicles, to maintain cellular organization and functionality, playing a crucial role in the overall metabolic efficiency of the cell.

How are ER proteins retrieved using a KDEL signal?

proteins containing a KDEL signal are recognized by KDEL receptors in the Golgi, which bind to them and facilitate their retrograde transport back to the endoplasmic reticulum (ER) through vesicular trafficking

What prevents KDEL receptor from binding in the wrong place?

pH, changes affinity of ER & binding to only bind the VTC & Golgito ensure selective retrieval of proteins. Golgi to

Where is protein sorting done?

Golgi apparatus, where proteins are modified, sorted, and packaged for transport to their final destinations within or outside the cell.

What is the Golgi apparatus?

3-20 cisternae/flattened sacs, 1-100s of Golgi per cell, modifies & sorts/make & leave using enzymes in each cisterna

cis face - ER & nucleus, trans face - plasma membrane, where vesicles bud off for transport.

Where does protein modification happen?

starts in ER & continues in Golgi, happen in sequences as they pass through each individual cisterna, 1 way direction

In what cases is traffic not unidirectional in the Golgi?

retrieval of proteins to the ER using KDEL receptors

What kinds of modifications are done in the Golgi?

modify sugars/glycosylation or removal , sort on trans side & send through, leaves looking different, sorted to be sent to:

• lysosomes

• plasma membrane

• secretory vesicle

How does golgi to lysosome trafficking work?

some things are made & immediately sent to lysosome, retrograde manner

What is the lysosome?

• uses internal membrane proteins, highly glycosylated to protect from proteases & acidic environment

• membrane contains transporters for recycling of monomers after macromolecule breakdown

• has a bilayer, has glycosylated proteins inside

How does the lysosome function?

• P-type pump/ATP-generated H+ gradient drives transport & maintains acidity, pumps from cytosol to lysosome, acidifies lysosome lumen

• transporters pump out to cytosol to recycle, why we make & break

• acid hydrolases for everything, function at an acidic pH 5 (nuclease, protease, glycosidase, lipase, phosphatase, sulfatase, phospholipases)

If a lysosome was to rupture & leak into the cytoplasm, will the cell die?

no, small & not a large concentration, acid hydrolases are turned off & need acidity to work

What are the ways that digestive material can fuse with lysosomes?

autophagy, receptor-mediated endocytosis, phagocytosis, pinocytosis

What is autophagy?

• self-eating, done during cellular distress when mitochondria wears out, injury, or low nutrients & needing energy by all cells

• originates in cytoplasm of cell, digests cytosol & worn-out organelles

• steps: nucleation, closure, autophagosome fusion with lysosomes, digestion

• induction is spontaneous, double membrane structure around target

What is receptor-mediated endocytosis?

endosomes from cell membrane contain molecules that must be processed, can fuse with lysosomes, outside in, done by all cells, clathrin mediated process, receptor binds to membrane & brings in

How does receptor-mediated endocytosis work?

receptors embedded in PM bind extracellular cargo & bring in, receptors are recycled through exocytosis back to PM, cargo sent to Golgi for sorting & lysosome

What is an example of receptor-mediated endocytosis?

cholesterol, same Clathrin process but receptor binds cholesterol & brings inside

Do ligand receptors cause cells to be vulnerable to infections?

yes, viruses can trick, mimic to bind to receptor & bring in with endocytosis, compete to bind

What is phagocytosis?

large particles & microorganisms from outside, done by WBCs, macrophages, neutrophils, not done by every cell

What is pinocytosis?

cellular drinking, endocytosis of small amounts of extracellular fluid, nonspecific targeting

What is LDL?

Low-Density Lipoprotein, a type of cholesterol carrier in the bloodstream that can contribute to plaque buildup in arteries.

What are lipoproteins?

Molecules made of proteins and lipids that transport fats and cholesterol through the bloodstream, since cholesterol can’t dissolve in blood, esterified molecules surrounded by proteins & lipid monolayer

What is the opposite of LDL & its difference?

High Density Lipoprotein, good cholesterol, less cholesterol per lipoprotein, scavenges lipoprotein from blood, package into protein compounds, LDL is bad in high amounts

If a cell wants cholesterol, what happens?

inserts receptors into PM, LDL binds receptors, internalized in clathrin-dependent mechanism