2.3: Vesicular Traffic to and From the Cell Surface

Secretion of Insulin in Pancreatic β-Cells: High Glucose

• when blood glucose levels rise, β-cells take up glucose

• glucose metabolism increases ATP production

• a high ATP/ADP ratio closes ATP-sensitive K⁺ channels → depolarization → Ca²⁺ influx

• Ca²⁺ triggers exocytosis of insulin-containing secretory granules

Secretion of Insulin in Pancreatic β-Cells: Retrograde Retrieval

• pancreatic β-cells package proinsulin → insulin into vesicles that bud from the trans Golgi network (TGN)

  • the early vesicles are called immature secretory granules

• when the vesicle first buds off, it contains extra “stuff” (insulin/proinsulin, membrane proteins, processing enzymes, etc.)

• retrograde retrieval: as vesicles mature, the cell retrieves unwanted proteins back to the Golgi (vesicle → Golgi)

  • as extra proteins leave, insulin becomes more concentrated

• the dense mature granules can rapidly release lots of insulin

  • they dock at the PM (inner surface) and are primed for rapid release (exocytosis)

Clathrin Vesicles

• bud from PM → endosomes (endocytosis)

• trans-Golgi network → endosomes/lysosomes

• specialize in cargo selection 

All Membrane-Bound Compartments are Connected

• proteins are successively modified as they pass thru a series of compartments

• some vesicles select cargo molecules and move them to the next compartment

• some vesicles retrieve escaped proteins and return them to a previous compartment

• the biosynthetic secretory is a continuous flow of material

Big Picture of Vesicles

Clathrin Triskelion

• clathrin is the main structural protein that forms the outer shell of the vesicle during endocytosis or TGN sorting

• forms a 3-legged structure (triskelion): has 3 heavy and 3 light chains

• the shape allows it to link to other triskelions at flexible joints

• isolated triskelions spontaneously self-assemble into a polyhedral cage

  • the cage bends the membrane and gives vesicles their shape 

Clathrin Vesicle Formation: Step 1

• adaptor proteins (AP) form a discrete second layer b/w the clathrin cage and the membrane

• AP complexes bind transmembrane cargo receptors (eg. LDL receptor), which bind soluble cargo inside the lumen

  • so AP complexes select which cargo gets packaged

  • once adaptors bind receptors, clathrin triskelia assemble on them; clathrin only binds via adaptor proteins (can’t bind membrane directly)

• there are several types of adaptor proteins, each specitfic for a subset of receptors

• Arf is a small GTPase that helps recruit AP complexes

  • belongs to family of small monomeric GTPase

• as more clathrin triskelia assemble, the membrane begins to curve

Clathrin Vesicle Formation: Step 1 Figure

Different AP Complexes

• AP1: TGN to endosomes

• AP2: endocytosis from the plasma membrane (bud inward for outside material)

• AP3: lysosome-related organelles

• AP4: specialized pathways

Clathrin Vesicle Formation: Step 2 Fission and Uncoating

• dynamin forms a ring around the neck of a budding vesicle

• dynamin is a soluble cystolic protein that contains a GTPase domain, which regulates the rate of pinching off

• dynamin uses GTP→GDP to change shape to tighten the ring

• as dynamin tightens, the inner leaflets of the pinched membrane come close enough to fuse, separating the vesicle from the membrane

• dynamin recruits other proteins at the budding vesicle to help bend the patch of membrane (distort the bilayer structure, change lipid composition)

• once released from the membrane, the vesicle rapidly loses its clathrin coat under the action of ARF GTPase

• the clathrin monomers and AP proteins are recycled

Clathrin Vesicle Formation: Step 2 Fission and Uncoating FIGURE

Clathrin Vesicle Formation: Step 3 Recruitment of Rab GTPase

• Rab GTPases are membrane-bound proteins

• Rab proteins bind to Rab effectors on the target membrane, pulling the vesicle close for fusion

  • some Rab effectors are tethering proteins that can extend 200nm above the membrane surface

• PIP (inositol lipids) are important for the selective distribution

Clathrin Vesicle Formation: Step 3 Recruitment of Rab GTPase FIGURE

Rab Proteins

• the largest subfamily of small GTPase (60 members)

• the selective distribution of Rab proteins at the surface of the vesicles guides vesicular 

PIPs + Rab GEFs Control Rab Binding Specificity

• different organelles have distinct PIP compositions; the specific lipid composition helps recruit the right Rab proteins

• lipid kinases and phosphatases control PIP levels (located on cystolic face, some integral, some peripheral)

  • kinases: add phosphate groups

  • phosphatases: remove phosphate groups

• RAB GEFs activate Rab GTPases, then Rab-GTP can bind specifically to membranes with the correct PIP signature

  • ensures vesicle targeting specificity

PIPs + Rab GEFs Control Rab Binding Specificity

Different PIPs for Different Organelles

• early endosome: PI3P

• PM: PI(4,5)P₂, PI(3,4)P₂, PI(3,4,5)P₃

• late endosomes, lysosomes: PI(3,5)P₂

• Golgi: PI4P

Forces Applied by SNAREs

• SNARE motif: ~60-70 a.a’s with heptad repeats (a repeating pattern of 7 amino acids that allow coiled- coil formation, ⍺-helices wind around each other like the strands of a rope)

• zipper model: a v-SNARE (on vesicle) and t-SNAREs (on target membrane) come together

  • they start pairing from the N-terminal ends (far from the membrane) and zipper toward the C-terminal ends

  • the zippering process pulls the the two membranes together

• membranes naturally repel each other, SNARE zippering overcomes these repulsive forces

  • when membranes get to ~1nm apart, spontaneous fusion can occur

Forces Applied by SNAREs: Energy

• SNARE complex formation releases a lot of energy

• strong enough to:

  • displace water b/w membranes

  • deform the lipid bilayers

  • form a hemifusion stalk

  • drive full fusion pore opening

Forces Applied by SNAREs: Figure

Clathrin Vesicle Formation: Step 5 Recycling SNAREs

• after fusion, the v-SNARE and t-SNAREs are now stuck tgt in. a very stable 4-helix bundle

• the SNAREs must be pulled apart so each one can be reused in another round of vesicle fusion

  • disassembly mediated by NSF (ATPase) and ⍺-SNAP (binds to SNAREs and recruits NSF) proteins

• NSF uses energy from ATP hydrolysis to forcibly unwind the SNARE complex

• v-SNAREs are then returned to the appropriate membrane and t-SNAREs remain at their membrane

NSF

• N-ethylmaleimide-sensitive factor

• uses multiple cycles of ATP hydrolysis to pry apart SNAREs

• hexamer of identical subunits (termed AAA ATPase) that associates with ⍺-snap (soluble NSF attachment protein)

NEM

• N-ethylmaleimide

• blocks NSF function 

• reacts with free SH group on cysteine residues

SNAREs Disassembly Mechanism: “Socket and Wrench”

• ⍺-SNAP is the socket that attaches onto the assembled SNARE complex

  • wraps around the complex and creates surface for NSF

• NSF is the wrench that clamps onto the socket and turns (using ATP)

• together, they produce mechanical force that unwinds the extremely stable helix bundle (V-SNARE and T-SNARE after membrane fusion)

SNAREs Disassembly Mechanism: “Socket and Wrench” FIGURE

Botulinic Toxin from Clostridium botulinum

• botulinum toxin and tetanus toxin are neurotoxins produced by Clotridium species that cleave SNARE proteins 

  • these toxins block synaptic transmissions

  • if SNAREs are cut → synaptic vesicles cannot fuse → no neurotransmitter release

• botulinum toxin blocks acetylcholine release at neuromuscular junctions → muscles cannot contract

  • botox destroys SNAREs preventing muscular contraction

• tetanus toxin blocks inhibitory neurons in the central neural system → constant muscle contraction

  • LD₅₀ ~ 1ng/kg (extremely potent), making it deadly

SNARE Complex

• formed by four ⍺-helices contributed by synaptobrevin, syntaxin and SNAP-25

• these provide the mechanical force to bring membranes together

Calcium Binding to Synaptotagmin Triggers Fusion

• synaptotagomin: a Ca²⁺ sensor embedded in the synaptic vesicle and controls whether fusion goes forward

• before Ca²⁺ arrives, synaptotagmin acts as a fusion clamp,

  • preventing premature fusion when vesicles are docked and primed, and the SNARE complex is partially zippered

• synaptotagmin binding to the SNARE complex causes the fusion clamp to tighten further and creates additional disturbance in the bilayer

• when Ca²⁺ enters the neuron, Ca²⁺ binds synaptotagmin

  • Ca²⁺-bound synaptotagmin releases the “clamp,” accelerates SNARE zippering, and triggers immediate vesicle fusion → neurotransmitter release

How the Spike Protein Works: SARS-CoV

• mediates viral entry and consists of 2 subunits

• S1 domain: forms the outer portion of the ectodomain, contains the receptor-binding domain

  • responsible for recognition and binding to the host cell receptor (Ace-2)

• S2 domain: responsible for fusion,

  • contains the putative fusion peptide (inserts into the host membrane after activation)

  • and the heptad repeat HR1 and HR2, forming a six-helix bundle that pulls viral and host membranes together

• activation by host protease TMPRSS2 (transmembrane serine protease), cleaves the spike protein at the S1/S2 site, priming the spike protein and exposing the fusion peptide → S2 undergoes conformational change that drives membrane fusion 

COPII Vesicles

• anterograde (ER→Golgi)

• carry newly synthesized proteins toward the Golgi

• COPII vesicle formation looks similar to clathrin coat assembly/disassembly

COPII Vesicle Formation

• SAR1-GTP inserts into the membrane and recruits the inner coat proteins Sec23/24

  • clathrin system: ARF1-GTP

• cargo selection: Sec24 recognizes cargo sorting signals in transmembrane cargo receptors

  • clathrin: AP (adaptor proteins)

• coat assembly + bud formation: Sec23/Sec24 inner coat recruits Sec13/Sec31 outer coat → cage forms

  • clathrin: AP proteins recruit triskelia

• fission is less dependent on dynamin-like protein but still involves curvature forces driven by the coat

• uncoating: Sar1 hydrolyzes GTP → coat disassembles after vesicle buds

  • clathrin: ARF-GTP hydrolysis

COPII Vesicles FIGURE

Fusion of COPII vesicles to form the ERGIC(ER-Golgi Intermediate Compartment)

• what happens after COPII uncoat

• uses the same Rab+SNARE machinery as clathrin

• COPII vesicles bud from the ER, but do not fuse directly with the Golgi, instead they first fuse with each other to form a collection of tubules and vesicles near the ER

  • the ER-Golgi Intermediate Compartment (ERGIC)

• ERGIC moves toward the Golgi, then fuses with cis-Golgi

COPI Vesicles

• retrograde (Golgi → ER, or earlier Golgi)

• return escaped ER-resident proteins (eg. BiP or ER-resident proteins)

• recycle Golgi enzymes, leaving compartments organized

Retrieval of ER-resident proteins

• resident ER membrane proteins contain signals that bind directly to COPI coats (KKXX at C-terminus)

• soluble ER resident proteins (e.g BiP) contain KDEL signal (Lys-Asp-Glu-Leu)

  • they bind to KDEL receptor

  • the affinity of the KDEL receptor depends on the environment (pH sensitive)

• the KDEL receptors sits in the Golgi membrane and bind in acidic Golgi pH, releases them in neutral ER pH

  • this prevents the KDEL sequence from interacting with the KDEL receptor in the ER

pH Controls the Affinity of KDEL Signal Figure

Golgi Apparatus

• allows for post-translational modifications (glycosylation, phosophorylation, sulfation)

• each Golgi stack has 2 distinct faces: a cis face (entry) and a trans face (exit)

• each stack (cisterna) contains a characteristic set of processing enzymes

• the Golgi apparatus generates heterogenous oligosaccharide structures, and complex oligosaccharides are added to proteins in the Golgi

  • the human genome encodes many different Golgi glycosyl transferases

• the Golgi resident proteins (glycosidases and glycosyl tranferases) are all membrane-bound

  • this way the retrieval is facilitated via the COPI mechanism

The Lysosome

• membrane-bound organelle in the cytoplasm of eukaryotic cells containing degradative, hydrolytic enzymes

  • the lysosomes membrane is highly glycosylated (needs to protected from the proteases and lipases)

• sites of intracellular digestion, serve to digest macromolecules

• about 40 types: proteases, nucleases, glycosidases, lipases, phosphatases, sulfatases

• proteins are synthesized in inactive form (proenzyme) and require an acidic environment for activation

• vacuole H⁺ ATPase uses the energy of ATP to pump H⁺ into the lysosome

• the H⁺ gradient provides energy for the transport of metabolites out of the organelle

More Lysosome Figures

Delivery of Material to Lysosome: Intracellular Traffic 

Delivery of enzymes from the Golgi

• the enzymes are made in the ER, processed in the Golgi and then tagged with mannose-6-phosphate (M6P)

• M6P receptors in the trans-Golgi network package them into vesicles heading toward endosomes → lysosomes

Delivery of cargo from endocytosis

• macromolecules (eg. LDL, proteins) enter an early endosomes thru endocytosis

• as the compartment acidifies, the early endosome becomes a late endosomes then matures into lysosomes

  • involves increasing acidity, acquisition of lysosomal enzymes from Golgi, recycling of endosomal membrane proteins back to the PM or TGN

Delivery of Material to Lysosome: Autophagy

Cells digest their own organelles or parts of the cytoplasm

• damaged organelles (eg. mitochondria every 10 days in liver cells, bulk cytoplasm (during starvation)

• a double-membrane structure forms around the material to form autophagosome, which fuses with lysosome/ late endosome

• contents are degraded and the breakdown products (a.a’s, lipids, sugars) are reused 

  • Metabolites derived from the digestion of the captured material help the cell survive

Delivery of Material to Lysosome: Phagocytosis

• for large particles and microorganisms

• primarily occurs in macrophages and neutrophils

• cell engulfs large particles/objects to form a phagosome

• phagosome fuses with a lysosome → particle is digested

The M6P Signal binds to M6P Receptor in the Golgi

• the M6P-tagged hydrolase binds to the receptor in the Golgi → packed in clathrin-coated vesicles for transport to endosomes/lysosomes

• the vesicle fuses with the lysosome and the low pH in the lysosome cause the hydrolase to release from the receptor 

• an acid phosphatase destroys M6P, prevents the enzyme from rebinding the receptor, keeping it in the lysosome

• M6P receptors are retrieved into coated transport vesicles

Lysosomal Storage Disease

• autosomal recessive disorder, no cure currently

• the enzyme GIcNAc-phosphotransferase (located in the cis-Golgi network) is defective or absent

  • this enzyme is required to add the M6P tag onto lysosomal hydrolases

• if M6P is not added, lysosomal hydrolases cannot bind to M6P receptors in the TGN → do not enter vesicles destined for lysosomes

  • instead follow default secretion pathway and are secreted outside the cell

• undigested substrates accumulate in lysosomes, resulting in inclusion cells (composed of carbohydrates, lipids, other materials that should be degraded)

Lysosomal Storage Disease: Symptoms

• Developmental delay

• Skeletal abnormalities

• Organ dysfunction

• Shortened lifespan