1. Chapter 14 – Vesicular Traffic, Secretion, and Endocytosis
Clathrin-coated vesicle formation:
AP2 complexes (green) –
Recruited to the inner surface of a membrane.
Capture three-legged clathrin triskelions.
Underlying membrane curvature increases as assembly of the clathrin-AP2 lattice proceeds.
Scission from membrane yields a coated vesicle.

2. Chapter 14 – Vesicular Traffic, Secretion, and Endocytosis
14.1 Techniques for Studying the Secretory Pathway 14.2 Molecular Mechanisms of Vesicle Budding and Fusion 14.3 Early Stages of the Secretory Pathway 14.4 Later Stages of the Secretory Pathway 14.5 Receptor-Mediated Endocytosis 14.6 Directing Membrane Proteins and Cytosolic Materials to the Lysosome

3. Vesicular Traffic, Secretion, and Endocytosis
14.1 Techniques for Studying the Secretory Pathway
Various labels enable tracking protein movement through the secretory pathway to different destinations.
Components required for intracellular protein trafficking have been identified by analysis of yeast temperature-sensitive secretory (sec) mutants.
Cell-free assays for intercompartmental protein transport have defined individual steps of the secretory pathway.

4. FIGURE 14-1 Overview of the secretory and endocytic pathways of protein sorting.
Secretory and endocytic pathway protein trafficking – unifying principle: transport vesicles transport membrane and soluble proteins from one membrane-bounded compartment to another
Transport vesicles:
Collect cargo proteins in membrane budding from a donor compartment
Deliver cargo proteins to the next compartment by fusing with the target membrane
Secretory pathway:
Distribution of soluble and membrane proteins synthesized by the rough ER to final destinations at the cell surface (including secretion) or in lysosomes
Two stages –
Stage 1: Rough endoplasmic reticulum –
Step 1: Synthesis of proteins bearing an ER signal/targeting sequence – cotranslational insertion of newly made polypeptide chains into the ER membrane or across it into the ER lumen
Stage 2: Protein trafficking –
Step 2: Proteins packaged into transport vesicles that bud from the ER and fuse together to form a new cis-Golgi cisternae
Step 3: ER enzymes or structural proteins –
Retained in the ER
Retrieved to the ER by vesicles that bud from the cis-Golgi and fuse with the ER
Step 4: Each cis-Golgi cisterna and contents moves from the cis to the trans face of the Golgi complex by nonvesicular cisternal maturation.
Step 5: Retrograde transport vesicles move Golgi-resident proteins to the previous Golgi compartment.
Step 6: Constitutive secretion (all cells) – transport vesicles move continuously and fuse with the plasma membrane
Soluble proteins are continuously secreted.
Membrane proteins become plasma membrane proteins.
Step 7: Regulated secretion (certain cell types) –
Proteins accumulated and stored in regulated secretory vesicles
Vesicles fuse with plasma membrane and secrete proteins only when cell receives a neuronal or hormonal signal secretion signal.
Step 8: Lysosome-destined membrane and soluble proteins – transported in vesicles that bud from the trans-Golgi and fuse with the late endosome for delivery to a lysosome
Endocytic pathway:
Step 9: Vesicles budding from the plasma membrane take up soluble extracellular proteins and deliver them to lysosomes via late endosomes.

5. EXPERIMENTAL FIGURE 14-2 Protein transport through the secretory pathway can be visualized by fluorescence microscopy of cells producing a GFP-tagged membrane protein.
Viral membrane glycoproteins:
Synthesized and inserted into the host cell membrane by the constitutive secretory pathway
Useful as an exogenous experimental probe to investigate membrane protein trafficking
Experiment:
Labeled plasma membrane protein – VSV virus G coat protein-GFP protein hybrid
Cultured cells – transfected with VSV G protein temperature-sensitive mutant-GFP protein hybrid gene
40°C – mutant VSV G-GFP is unable to fold and retained in the ER
(a) Shift to 32°C at 0 min – VSV G-GFP folds properly
40 min – most VSV G-GFP concentrated in the Golgi
180 min – most VSV G-GFP has integrated into and is diffusing within the plasma membrane
(b) Amount of VSVG-GFP in the endoplasmic reticulum (ER), Golgi, and plasma membrane (PM) at times after the shift to the permissive temperature
Kinetics of transport from ER-Golgi-plasma membrane
(Decrease in total GFP fluorescence at later times results from slow inactivation of GFP fluorescence.)

6. EXPERIMENTAL FIGURE 14-3 Transport of a membrane glycoprotein from the ER to the Golgi can be assayed based on sensitivity to cleavage by endoglycosidase D.
Detecting secretory protein carbohydrate modifications by compartment-specific enzymes elucidates trafficking through different compartments.
Experiment:
Temperature-sensitive VSV G protein – labeled with a pulse of radioactive amino acid during synthesis in cells
Split cells into two groups –
Group 1 – Control cells maintained at 40°C – misfolded protein retained in ER
Group 2 – Experimental cells shifted to 32°C – protein moves through secretory pathway
Time points – VSV G protein extracted and treated with endoglycosidase D
(a) Protein movement to cis-Golgi and modification:
Protein retained in the ER – not modified
Protein moved from the ER to the cis-Golgi – core oligosaccharide Man8(GlcNAc)2 is trimmed to Man5(GlcNAc)2 by cis-Golgi resident enzymes
Endoglycosidase D – cleaves the oligosaccharide chains from proteins processed in the cis-Golgi, but not from proteins retained in the ER
(b) SDS-polyacrylamide gel electrophoresis of the digestion mixtures (Autoradiography only shows labeled VSV G protein in gel.):
(ER) resistant – uncleaved (larger-slower-migrating)
(cis-Golgi) sensitive – cleaved (smaller-faster-migrating)
5 min – most protein resistant to EndoD cleavage – not yet moved from ER to cis-Golgi ER
60 min – most protein cleaved by EndoD cleavage – moved to and modified in the cis-Golgi
(c) Percentage of EndoD-sensitive VSV G protein over time:
Derived from electrophoretic data
40°C – little increase n sensitive protein – misfolded proteins retained in ER
32°C – most protein moved from ER to cis-Golgi and modified by 30–40 min

7. EXPERIMENTAL FIGURE 14-4 Phenotypes of yeast sec mutants identified five stages in the secretory pathway.
Yeast temperature-sensitive mutants define major stages and individual components involved in targeting and vesicular transport.
Five classes of temperature-sensitive mutants:
Based on the site where newly made secretory proteins (red dots) accumulate when cells are shifted from the permissive temperature to the higher, nonpermissive one
Cytosol
Rough ER
Vesicles transporting proteins from the ER to the Golgi complex
Golgi cisternae
Constitutive secretory vesicles
Double mutant analysis – defines order of steps.
Mutations in both class B and class D functions – proteins accumulate in the rough ER, not in the Golgi cisternae

8. EXPERIMENTAL FIGURE 14-5 A cell-free assay demonstrates protein transport from one Golgi cisterna to another.
Cell-free transport assays – biochemical dissection of secretory pathway individual steps
Experiment:
VSV virus infection of cultured mutant cells lacking one of the Golgi enzymes that modify N-linked oligosaccharide chains –fate of the VSV G protein.
(a) VSV G protein modification in Golgi of intact wild-type and mutant fibroblast cells
Wild-type cells:
medial-Golgi – N-acetylglucosamine transferase I adds one N-acetylglucosamine to N-linked oligosaccharides
trans-Golgi – N-linked oligosaccharides further modified to more complex carbohydrate
Mutant cells – lack medial-Golgi N-acetylglucosamine transferase I:
medial-Golgi – no addition of N-acetylglucosamine
trans-Golgi – no further modification to complex carbohydrate
G protein reaches the cell surface with a simpler high-mannose oligosaccharide containing only two N-acetylglucosamine and five mannose residues.
(b) Golgi isolated from VSV-infected mutant cells and noninfected wild-type cells:
Mutant cell Golgi – contains VSV G protein
Wild-type cell Golgi – no VSV G protein
Golgi mixed together
Result: Some VSV G protein modified by wild-type medial-Golgi N-acetylglucosamine transferase I
Conclusion: N-acetylglucosamine transferase I – moved by transport vesicles from the wild-type medial-Golgi cisternae to the mutant cis-Golgi during cisternal maturation process.

9. Vesicular Traffic, Secretion, and Endocytosis
14.2 Molecular Mechanisms of Vesicle Budding and Fusion
Three types of coated vesicles mediate protein transport through different pathways.
Small GTPase proteins direct coat protein polymerization on donor membranes to pinch off vesicles carrying different cargoes.
Coat shedding exposes Rab and SNARE proteins that target vesicles for fusion with specific target membranes.

10. FIGURE 14-6 Overview of vesicle budding and fusion with a target membrane.
Vesicles:
Bud from the a donor membrane
Fuse with specific target membrane
Assembly of a protein coat drives vesicle formation and selection of specific cargo molecules.
(a) Vesicle budding from donor membrane:
Recruitment of GTP-binding G proteins to a region of donor membrane
Cytosolic coat protein complexes bind to the cytosolic domain of membrane cargo proteins.
Coat binding evaginates the membrane – typical transport vesicle; ~50 nm in diameter
Some cargo proteins act as receptors to bind soluble proteins in the lumen and capture them into the budding vesicle.
Donor membrane–specific SNARE proteins (vesicle-SNARES; v-SNARES) captured in budding vesicle membrane.
Donor membrane fusion – pinches off coated vesicle
Coated vesicle uncoated in cytosol – uncovers projecting v-SNARES for fusion targeting
(b) Vesicle fusion with target membrane:
Targeting – interaction of specific v-SNARE with specific target membrane t-SNARES
Fusion process (Fig 14-10)

11. Table 14-1 Coated Vesicles Involved in Protein Trafficking
Three major types of transport-specific coated vesicles: each with a different type of protein coat formed by reversible polymerization of a distinct set of coat and G protein subunits.
A conserved set of monomeric GTPase switch proteins controls the assembly of different vesicle coats.

12. EXPERIMENTAL FIGURE 14-7 Vesicle buds can be visualized during in vitro budding reactions.
Experiment: Incubate isolated ER vesicles or artificial phospholipid vesicles (liposomes) with purified COPII coat components.
Result (EM image): Coat protein polymerization on the vesicle surface – induces emergence of highly curved buds

13. FIGURE 14-8 Model for the role of Sar1 in the assembly and disassembly of the COPII coat.
Sar1/ARF: cycle of GEF-activated GTP binding and GAP-activated GTP-hydrolysis controls assembly and disassembly of vesicle coats.
Step 1:
Sar-GDP interacts with the ER integral membrane protein Sec12.
Sec12 GEF activity – stimulates Sar1 GTP for GDP exchange
Sar1 conformational change – integrates its N-terminal amphipathic helix into the ER membrane outer leaflet
Step 2:
Sar1-GTP recruits the Sec23/Sec24 coat protein complex.
Sec23/Sec24 – binds specific short sequences (sorting signals) in membrane cargo protein cytosolic domains
Sec13/Sec31 coat complex assembles into coat (not shown) – completes coat assembly
Coat assembly – curves and pinches off ER membrane region as a COPII-coated vesicle containing cargo proteins and v-SNARES
Step 3: Sec23 GAP activity – stimulates Sar1 GTP hydrolysis to GDP
Step 4: Release of Sar1-GDP from the vesicle membrane – causes disassembly of the coat

14. EXPERIMENTAL FIGURE 14-9 Coated vesicles accumulate during in vitro budding reactions in the presence of a nonhydrolyzable analog of GTP.
Experiment – isolated Golgi membranes + cytosolic extract containing COPI coat proteins:
Vesicles form and bud off from the Golgi membranes.
+ Nonhydrolyzable GTP analog – inhibits ARF GTP hydrolysis (similar results with ARF mutated to only lose GTPase activity)
Vesicles form and pinch off donor membrane.
Coat fails to disassemble following vesicle release from membrane.
EM – purified COPI-coated vesicles (further analyzed to investigate COPI components and properties)

15. Table 14-2 Known Sorting Signals That Direct Proteins to Specific Transport Vesicles
Targeting sequences on the cytosolic regions of cargo proteins bind to specific coat proteins.

16. FIGURE 14-10 Model for docking and fusion of transport vesicles with their target membranes.
Rab GTPases control docking of vesicles on target membranes.
(a) Fusion of secretory vesicles with the plasma membrane – similar mechanism mediates all vesicle-fusion events:
Step 1: Transport vesicle docking on appropriate target membrane – Rab protein tethered via a lipid anchor to a secretory vesicle binds to an effector protein complex on the target (plasma) membrane.
Step 2: v-SNARE protein (VAMP) forms stable coiled-coil interaction with cognate t-SNAREs cytosolic domains (syntaxin and SNAP-25) –
SNARE complexes – hold vesicle close to the target membrane
Step 3: Fusion of the two membrane – drives dissociation of the SNARE complexes, freeing SNARE proteins for another round of vesicle fusion
Rab-GTP hydrolyzed to Rab-GDP – dissociates Rab from the Rab effector (not shown)
(b) v-SNARE–t-SNARE complex:
Four long α helices, two from SNAP-25 and one each from syntaxin and VAMP, form numerous noncovalent interactions to form four-helix coiled-coil.

17. Vesicular Traffic, Secretion, and Endocytosis
14.3 Early Stages of the Secretory Pathway
COPII-coated vesicles transport newly synthesized proteins containing Golgi-targeting sequences in their cytosolic domain or bound to such proteins from the rough ER to the cis-Golgi (anterograde direction).
COPI-coated vesicles transport vesicles carrying ER/Golgi-resident proteins in the retrograde direction, which supports Golgi cisternal maturation.

18. FIGURE 14-11 Vesicle-mediated protein trafficking between the ER and cis-Golgi.
Vesicle transport between ER and cis-Golgi – initial transport stage of secretory pathway
Forward (anterograde) transport – COPII vesicle-mediated ER to cis-Golgi transport
Cargo – newly synthesized proteins
Step 1:
COPII coat assembles on ER membrane to form vesicles containing –
v-SNAREs (orange)
Membrane cargo proteins (blue)
Soluble cargo proteins (magenta) bound to appropriate receptors in the membrane of budding vesicles.
COPII-coated vesicle pinches off ER membrane.
COPII pinches off coated vesicle.
Step 2: COPII coat disassembly exposes v-SNARE proteins on the vesicle surface for fusion targeting.
Step 3:
Rab-effector interaction promotes association of v-SNAREs with t-SNARES on cis-Golgi membrane.
Membrane fusion – releases vesicle contents into the cis-Golgi compartment and membrane
Reverse (retrograde) transport – COPI vesicle-mediated cis-Golgi to ER transport
Cargo – recycles the membrane bilayer, v-SNAREs, and missorted ER-resident proteins (not shown)
Step 4: ARF-GTP recruits COPI coat.
Step 5: COPI-coated vesicles pinch off cis-Golgi membrane.
Step 6: cis-Golgi v-SNARE interaction with ER t-SNARE mediates vesicle fusion with the ER membrane to deliver cargo.

19. FIGURE Three-dimensional structure of the ternary complex comprising the COPII coat proteins Sec23 and Sec24 and Sar1·GTP.
COPII vesicle recruitment of cargo proteins:
Sar1-GTP recruits Sec23/Sec24 to the ER membrane.
Sec24 interaction with cytosolic domain di-acidic targeting signal (purple) recruits cargo protein into the vesicle membrane.
Soluble cargo proteins interact with luminal regions of membrane cargo proteins.
Human disease – Cystic Fibrosis:
Cells in lungs and other organs missing CFTR plasma membrane Cl– transporter.
CFTR contains a di-acidic sorting signal – delivered to plasma membrane by the constitutive secretory pathway
ΔF508 mutation – major cause of CF
Misfolded cytosolic domain prevents CFTR recruitment into ER COPII vesicles for ER-Golgi transport.
CFTR eventually degraded in ER.

20. FIGURE 14-13 Role of the KDEL receptor in retrieval of ER-resident luminal proteins from the Golgi.
cis-Golgi network – ER-to-Golgi intermediate sorting compartment
Soluble ER luminal resident proteins:
Function in the ER to modify newly synthesized proteins
Several types contain a C-terminal KDEL (Lys-Asp-Glu-Leu) ER-targeting sequence.
May be missorted to the cis-Golgi network
Step 1: Nonspecifically incorporated in a COPII vesicle fluid phase
Step 2: Fusion of the vesicle delivers the ER resident protein to the cis-Golgi network.
cis-Golgi network to ER retrieval mechanism:
cis-Golgi network –
Membrane contains KDEL receptors.
Acidic pH – promotes receptor-KDEL interaction
Retrieval from cis-Golgi network to ER –
Step 3: ER resident-KDEL protein binding to a KDEL receptor incorporates the ER resident protein into a COPI-vesicle.
Step 4: COPI-vesicle fusion with the ER – returns the ER resident protein to the ER
Higher pH of ER – dissociates ER resident KDEL protein from the KDEL receptor (recycled back to the cis-Golgi network via COPII-vesicles)
Retrieval system prevents depletion of ER luminal proteins needed for proper folding of newly made secretory proteins.
Addition of KDEL sequence to C-terminus of protein normally secreted – becomes localized in ER

21. FIGURE Processing of N-linked oligosaccharide chains on glycoproteins within cis-, medial-, and trans-Golgi cisternae in vertebrate cells.
Golgi complex – three biochemical processing compartments contain different enzymes:
Anterograde transport through the three Golgi processing compartments occurs by cisternal maturation.
Specific transferase enzymes – add sugars to oligosaccharides from sugar nucleotide precursors imported from the cytosol
cis-Golgi:
Step 1: three mannose residues removed
medial-Golgi:
Steps 2 and 4: three N-acetylglucosamine (GlcNAc) residues added.
Step 3: two more mannose residues removed
Step 5: single fucose added
trans-Golgi:
Step 6: three galactose residues added
Step 7: N-acetylneuraminic acid residue added to each of the galactose residues

22. EXPERIMENTAL FIGURE Electron Micrograph of the Golgi complex in a pancreatic acinar cell reveals secretory and retrograde transport vesicles.
Pancreatic acinar cells –
Synthesize digestive enzymes – stored in regulated secretory granules
Secrete the digestive enzymes when signaled by feeding response
Rough ER – buds vesicles to transport proteins to the cis-Golgi network
Cisternal progression –
Small vesicles interspersed among the Golgi cisternae transport Golgi enzymes in the retrograde direction.
Vesicle fusion and delivery of Golgi enzymes converts one compartment to the next (cis to medial; medial to trans).
trans-Golgi network forms regulated secretory vesicles.

23. EXPERIMENTAL FIGURE Fluorescence-tagged fusion proteins demonstrate Golgi cisternal maturation in a live yeast cell.
Experiment – yeast cisternal maturation:
Yeast cells expressing:
cis-Golgi protein Vrg4 fused to GFP (green)
trans-Golgi protein Sec7 fused to DsRed (red)
Protein localizations imaged over time (min).
Results:
(top) – Both tagged proteins change localization over time.
(bottom) – Golgi cisterna maturing –
cis to medial maturation – compartment loses Vrg4
medial to trans maturation – compartment gains Sec7
medial compartment – contains both proteins for less than one minute
Conclusion: Vesicles budding off medial compartment remove Vrg4 and vesicles budded off trans compartment fusing with medial compartment deliver Sec7, as compartment progresses through the Golgi.

24. Vesicular Traffic, Secretion, and Endocytosis
14.4 Later Stages of the Secretory Pathway
The trans-Golgi network sorts proteins into vesicles targeted for different destinations.
Lysosomal enzymes bear M6P residues that are recognized by M6P-receptors and delivered by a clathrin-coated vesicle pathway to lysosomes.
Regulated secretory proteins are concentrated and stored until secretion is signaled; constitutively secreted proteins are continuously delivered to the plasma membrane.
Some proteins are processed into mature form after leaving the trans-Golgi network.

25. FIGURE 14-17 Vesicle-mediated protein trafficking from the trans-Golgi network.
Distal sorting compartment
Sorts proteins into five different types of vesicles for transport to the plasma membrane, endosomes, and lysosomes.
Five destinations:
(1) COPI vesicles: retrograde transport of Golgi enzymes to the trans- Golgi (cisternal progression process)
(2) AP complex vesicles (may have clathrin coat): transport lysosomal enzymes directly to lysosomes
(3) Clathrin-coated (+AP2) vesicles: transport lysosomal enzymes to late endosomes for eventual delivery to lysosomes
(4) Constitutive secretory vesicles (unknown coat):
Transport constitutively secreted proteins and plasma membrane proteins to the plasma membrane.
Cargo proteins include ECM proteins, blood proteins, immunoglobulins.
(5) Regulated secretory vesicles (unknown coat):
Store and process secreted proteins until signaled to fuse with the plasma membrane to secrete the proteins
Cargo proteins include digestive enzymes and peptide hormones

26. FIGURE 14-18 Structure of clathrin coats.
Clathrin-coated vesicles:
Bud from the trans-Golgi and plasma membranes
Clathrin coat interaction with a membrane – mediated by AP (adapter protein) complexes
AP complex:
One copy each of four different adapter subunit proteins
Interacts with clathrin HC globular domain and specific cytoplasmic domain sorting signal motifs of cargo membrane proteins
(a) Clathrin subunit triskelion structure:
Three long bent heavy chains – one end associates with two others to form triskelion
Three light chains – associated with HCs near vertex
Intrinsic curvature due to the bend in the heavy chains
(b) Clathrin coats – (CryoEM reconstruction)
Assembled in vitro by mixing purified clathrin heavy and light chains with AP2 complexes in the absence of membranes
Composed of 36 triskelions – each associated with others through HC tail interactions
Contains hexagon and pentagon arrangements
Vesicle uncoating – clathrin coat depolymerization:
ARF GTP hydrolysis regulates timing.
Mediated by cytosolic Hsp70 (chaperone, uncoating mechanism unknown) – activated by auxillin (co-chaperone), which stimulate Hsp70 ATP hydrolysis
Releases triskelions for reuse
Exposes v-SNAREs for fusion with target membranes

27. FIGURE 14-19 Model for dynamin-mediated pinching off of clathrin-coated vesicles.
Required for pinching off of clathrin-coated vesicles
Polymerizes around vesicle neck
Hydrolyzes GTP – conformational change coupled to membrane fusion and vesicle release from the donor membrane

28. EXPERIMENTAL FIGURE GTP hydrolysis by dynamin is required for the pinching off of clathrin-coated vesicles in cell-free extracts.
Experiment:
Nerve terminals –
Plasma membrane lysed with distilled water
Incubated with GTP-γ-S nonhydrolyzable analogue of GTP
Sectioned preparation – treated with gold-tagged anti-dynamin antibody and imaged with EM
Result: Polymerized dynamin lines the long neck of a clathrin/AP-coated bud.
(Extensive polymerization of dynamin in GTP-γ-S probably does not occur during the normal budding process.)
(Similar results with dynamin GTPase mutants.)
Conclusions: Dynamin GTP hydrolysis –
Not required for polymerization around bud neck
Required for pinching vesicle off donor membrane

29. FIGURE Formation of mannose 6-phosphate (M6P) residues that target soluble enzymes to lysosomes.
M6P residues direct newly synthesized lysosomal enzymes to lysosomes.
M6P targeting signal addition by two cis-Golgi-resident enzymes:
Step 1: cis-Golgi-localized N-acetylglucosamine (GlcNAc) phosphotransferase –
Recognizes specific protein sequences in newly synthesized lysosomal enzymes
Transfers a phosphorylated GlcNAc group to carbon 6 of one or more mannose residues
Step 2: Phosphodiesterase removes the GlcNAc group, leaving a 6-phosphorylated mannose residue on the lysosomal enzyme.

30. FIGURE Trafficking of soluble lysosomal enzymes from the trans-Golgi network and cell surface to lysosomes.
Mechanism:
Step 1: M6P receptors in the trans-Golgi membrane recruit M6P-lysosomal enzymes into clathrin/AP1-coated vesicles.
Step 2: Vesicle clathrin coat disassembles.
Step 2a: Coat proteins recycle to trans-Golgi membrane.
Step 3:
Vesicle fuses with late endosome compartment.
Acidic endosome pH – dissociates M6P-enzyme from receptor
Step 4: Late endosome fuses with a lysosome – delivers M6P-enzyme to targeted final destination
Step 4a: M6P receptors recycled – captured in vesicle targeted to trans-Golgi
Step 5: Some receptors are delivered to the cell surface.
Missorted M6P enzymes: imperfectly sorted enzymes – constitutively secreted (could cause extracellular damage)
Step 6: Plasma membrane M6P receptors capture missorted M6P-enzymes into clathrin-coated vesicles in the receptor-mediate endocytosis pathway.
Step 7: Coat disassembly forms uncoated endosome.
Step 8: Endosome fusion with late endosome and dissociation from receptor localizes M6P-enzyme for delivery to a lysosome.
Human I cell disease:
Lack of N-acetylglucosamine phosphotransferase
Lysosomal enzymes lack M6P targeting signal – constitutively secreted
Undigested lysosomal contents –
Continued accumulation forms “inclusions” – may eventually kill cell
Undegraded debris causes developmental, physiological, and neurological abnormalities.
In vitro “cure” – M6P-enzymes added to I-cell fibroblasts – internalized by endocytosis pathway and function on arrival in lysosomes

31. EXPERIMENTAL FIGURE Proteolytic cleavage of proinsulin occurs in secretory vesicles after they have budded from the trans-Golgi network.
Some proteins undergo proteolytic processing after leaving the trans-Golgi.
Experiment – insulin proteolytic processing in regulated secretory vesicles:
Insulin-secreting cells – stained with electron-opaque gold particles coated with –
(a) a monoclonal antibody that recognizes proinsulin, but not insulin (inset – proinsulin-rich secretory vesicle surrounded by a protein coat)
(b) a monoclonal antibody that recognizes insulin, but not proinsulin
Results:
Immature secretory vesicles –
Stain with the proinsulin antibody, but not with the insulin antibody
Contain diffuse protein aggregates that include proinsulin and other regulated-secreted proteins
Mature secretory vesicles –
Stain with insulin antibody, but not with proinsulin antibody
Accumulate a dense core of almost crystalline insulin
Conclusion: Proteolytic processing of proinsulin to insulin occurs in immature secretory vesicles during maturation to mature secretory vesicles.

32. FIGURE Proteolytic processing of proproteins in the constitutive and regulated secretory pathways.
Proproteins:
Matured into final form after leaving the trans-Golgi
Include soluble lysosomal enzymes; many membrane proteins, such as influenza hemagglutinin (HA); and secreted proteins such as serum albumin, insulin, glucagon, and the yeast α mating factor.
(a) Proalbumin processing – typical of proteins in constitutive secretory pathway:
Furin endoprotease – cleaves peptide at the C-terminal end of two consecutive amino acids
(b) Insulin processing – typical of proteins in regulated secretory pathway:
PC2 and PC3 endoproteases – cleave central region of insulin
Carboxypeptidase – sequentially cleaves two C-terminal basic amino acid residues
(Note: Insulin processed peptides – covalently linked by disulfide bonds)

33. FIGURE Sorting of proteins destined for the apical and basolateral plasma membranes of polarized cells.
Several pathways sort membrane proteins to apical or basolateral membrane regions of polarized cells with tight junctions.
Cultured polarized MDCK cells infected with both VSV and influenza viruses:
Influenza HA glycoprotein –
Only on the apical membrane (Influenza viruses bud from only the apical membrane.)
Sorted into vesicles from trans-Golgi targeted by specific Rab and v-SNARE for fusion with the apical membrane.
VSV G protein –
Contain cytosolic domain tyrosine-based motif and a di-leucine-based motif targeting sequences
Sorted into vesicles from trans-Golgi targeted by specific Rab and v-SNARE for fusion with the basolateral membrane
Only on the basolateral membrane (VSV viruses bud only from the basolateral membrane.)
Some cellular proteins – sorted similarly to only apical (including GPI proteins in some cell types) or basolateral membranes
Some hepatocyte apical membrane proteins –
Sorted initially to basolateral membrane
Selectively endocytosed into clathrin-coated vesicles and transcytosed through endosomes to the apical membrane

34. Vesicular Traffic, Secretion, and Endocytosis
14.5 Receptor-Mediated Endocytosis
Extracellular ligands bound to specific cell-surface receptors with cytoplasmic domain AP2-targeting sequences are internalized by clathrin-coated vesicles.
The endocytic pathway delivers some ligands (e.g., LDL particles) to lysosomes, where they are degraded.
The late endosome acidic environment dissociates most receptor-ligand complexes for receptor recycling to the plasma membrane and ligand degradation in lysosomes.
The iron endocytosis pathway releases Fe3+ in the late endosome but recycles the transferrin carrier proteins with the receptor to the plasma membrane .

35. EXPERIMENTAL FIGURE The initial stages of receptor-mediated endocytosis of low-density lipoprotein (LDL) particles are revealed by electron microscopy.
Receptor-mediated endocytosis (RME):
Used by cells to import specific macromolecules/complexes too large to be imported by membrane transporters
Uptake specificity – receptor-dependent
Uptake mechanism – ligand-receptor complexes incorporated into clathrin/AP2-coated vesicles
Experiment:
LDL particles (contain cholesterol) – labeled with electron-dense, iron-containing ferritin protein (visible with EM)
Cultured fibroblasts incubated with LDL-ferretin at 4°– LDL binds to LDL-receptors; endocytosis process is inhibited
Cells warmed to initiate endocytosis.
(a) LDL-LDL receptor complexes – cluster over invaginated clathrin-coated pit
(b) Pit containing LDL-LDL receptor complexes closes. (Dynamin associates with neck region.)
(c) Clathrin-coated vesicle containing LDL-LDL receptor complexes – free in cytoplasm
(d) LDL-LDL receptor complexes delivered to endosome. (6 minutes after internalization began)
RME receptors:
Some types cluster in clathrin-coated pits by cytoplasmic domain association with AP2 even in absence of ligand.
Others types diffuse freely in the plasma membrane until a ligand-induced conformational change associates them with AP2.
Two or more types of receptor-bound ligands, such as LDL and transferrin, can be present in the same coated pit/vesicle.

36. FIGURE 14-27 Model of low-density lipoprotein (LDL).
Cells take up lipids from the blood in the form of large, well-defined lipoprotein complexes.
All classes of lipoproteins have the same general structure:
Shell composed of apolipoprotein and a phospholipid monolayer (not bilayer) containing cholesterol
Hydrophobic core composed mostly of cholesteryl esters/triglycerides (with minor amounts of other neutral lipids [e.g., some vitamins])
LDL particle – contains only a single molecule of one type of apolipoprotein (ApoB) wrapped around the outside of the particle

37. EXPERIMENTAL FIGURE Pulse-chase experiment demonstrates precursor-product relations in cellular uptake of LDL.
Experiment:
LDL labeled with 125I
Cultured normal human skin fibroblasts – incubated with 125I-LDL for 2 hours at 4°C (pulse) – LDL binds to surface LDL receptors; not endocytosed
Unbound LDL – washed away
Shift to 37°C – activates RME (chase)
Results:
125I-LDL rapidly disappears from surface (binding) by RME.
125I-LDL in internal vesicles rises coincidently.
After a 15 min lag – 125I-LDL degradation in lysosomes increases.

38. FIGURE 14-29 Endocytic pathway for internalizing low-density lipoprotein (LDL).
Step 1: Cell-surface LDL receptor –
Extracellular domain – binds LDL particle ApoB protein tightly at extracellular neutral pH.
Cytosolic tail NPXY sorting signal – interaction with AP2 complex incorporates the LDL-LDL receptor complex into clathrin-coated pit
Step 2: Clathrin-coated pit containing receptor-LDL complexes –
Pinches off as clathrin-coated vesicle by dynamin-mediated mechanism
Clathrin disassembly uncoats vesicle.
Step 3: Vesicle fuses with endosome.
Endosome acidic pH – receptor conformational change releases LDL particle
Step 5: LDL receptor – recycled to the cell surface (LDL receptor round trip – 10–20 minutes)
Step 4: Late endosome fusion with lysosome –
LDL particle – broken down to constituent parts by lysosomal enzymes
LDL components recovered into cell as nutrients

39. FIGURE 14-30 Model for pH-dependent binding of LDL particles by the LDL receptor.
(a) LDL receptor at neutral pH (as at the cell surface):
Ligand binding arm seven cysteine-rich repeats (R1–R7) – tightly bind LDL apoB-100 (R4 and R5 – most critical for LDL binding)
(Note NPXY AP2-targeting sequence in receptor cytosolic domain.)
(b) LDL receptor at acidic pH (as in endosome):
β-propeller domain histidine residues – become protonated
Positively charged propeller domain binds negatively charged ligand-binding domain residues – causes release of the LDL particle
(b, bottom) LDL receptor at pH 5.3 structure – extensive hydrophobic and ionic interactions between the β propeller and R4 and R5 repeats
Human disease – Familial Hypercholesterolemia
Excessive circulating LDL causes cardiovascular disease.
LDL receptor mutations cause too little LDL uptake/clearance into cells (liver cells).
Several LDL receptor mutations including a single amino acid mutation in NPXY targeting sequence causes inefficient LDL receptor RME – FH disease.

40. FIGURE 14-31 The transferrin cycle operates in all growing mammalian cells.
RME endocytic pathway delivers iron to cells without dissociation of the transferrin–transferrin receptor complex in endosomes.
Transferrin protein:
Apotransferrin – no bound Fe3+
Ferrotransferrin – carries Fe3+ in blood
Binds to transferrin receptor
Uptake mechanism:
Step 1: Ferrotransferrin dimer with two Fe3+ binds tightly to transferrin receptor at the cell surface – ~pH 7.0.
Step 2: Transferrin receptor cytoplasmic tail interaction with an AP2 adapter complex – incorporates receptor-ligand complex into endocytic clathrin-coated vesicles
Step 3: Clathrin disassembly uncoats vesicle.
Step 4: Vesicle fuses with late endosome – acidic pH –
Dissociates Fe3+ from ferrotransferrin –
Fe3+ reduced to Fe2+ by endosome reductase
Transported from endosome to cytoplasm
Maintains apotransferrin-receptor complex
Step 5: Recycling of receptor-apotransferrin complex to the plasma membrane
Step 6: Extracellular neutral pH destabilizes complex – releases apotransferrin from receptor

41. Vesicular Traffic, Secretion, and Endocytosis
14.6 Directing Membrane Proteins and Cytosolic Materials to the Lysosome
Endocytosed membrane proteins targeted for degradation in the lysosome are incorporated into vesicles that bud into the interior of the endosome.
Cellular components (e.g., ESCRT) that mediate endosome membrane budding are used to pinch off enveloped viruses such as HIV from the plasma membrane of virus-infected cells.
Autophagy envelopes a region of cytoplasm or an organelle into a double-membrane autophagosome for delivery to a lysosome.

42. FIGURE 14-32 Delivery of plasma-membrane proteins to the lysosome interior for degradation.
Membrane proteins targeted for degradation – delivered to the lysosome lumen
Mechanism:
Step 1: Vesicles carrying newly synthesized lysosomal membrane proteins (green) from the trans-Golgi network – fuse with the late endosome
Step 2: Endosomes carrying endocytosed plasma-membrane proteins (blue) targeted for degradation – fuse with the late endosome
Step 3: Late endosome –
Plasma membrane proteins targeted for degradation – incorporated into vesicles that bud into the interior of the late endosome
Forms a multivesicular endosome
Step 4: Multivesicular endosome fuses with a lysosome –
Internal vesicles (containing targeted membrane proteins)– degraded
Lysosomal membrane proteins – not degraded

43. FIGURE 14-33 Model of the mechanism for formation of multivesicular endosomes.
Most proteins targeted for a multivesicular endosome degradation – tagged with ubiquitin at the plasma membrane, in the trans-Golgi network, or in the endosomal membrane
Multivesicular endosome formation mechanism:
Step 1: Ubiquitinylated Hrs protein on the endosomal membrane –
Directs loading of ubiquitinylated membrane cargo proteins (blue) into endosome vesicle buds
Recruits and assembles cytosolic ESCRT protein complexes on the endosome membrane
Step 2: ESCRT complexes – mediate the completion and pinching off of inwardly budding vesicles
Step 3: Vps4 ATP hydrolysis – drives disassembly/recycling of ESCRT complex proteins

44. FIGURE 14-34 Mechanism for budding of HIV from the plasma membrane.
Retrovirus (HIV) particle budding from the plasma membrane – exploits process for formation of multivesicular endosomes
(a) HIV particle budding mechanism:
~4000 virus Gag proteins – polymerize into a plasma membrane spherical shell projecting out from infected cell plasma membrane
Step 1: Virus core particle captured in bud
Step 2: Ubiquitinated Gag proteins function like Hrs protein – recruit ESCRT protein complexes to pinch off viral particle
Step 3: Vps4 disassembles ESCRT complex.
(b) HIV-infected wild-type cells: virus particles bud from the plasma membrane and are rapidly released into the extracellular space.
(c) HIV-infected cells lacking functional ESCRT Tsg101 protein:
Viral Gag proteins form dense virus-like structures unable to bud off membrane.
Virus-like structures form chains of incomplete viral buds still attached to the plasma membrane.

45. FIGURE 14-35 The autophagic pathway.
Autophagic pathway – delivers cytosolic proteins and organelles to lysosomes for degradation
Mechanism:
ATG proteins induce formation of cup-shaped membrane structure around –
(right) a portion of the cytosol
(left) an organelle such as a mitochondrion
Proteins involved include Atg 5, 8, 12, 16 – Atg 8 forms coat around autophagosome
Step 1: Continued membrane addition and fusion forms autophagosome – envelops contents in two complete membranes
Step 2: Fusion of the autophagosome outer membrane with the lysosome membrane –
Releases a single-membrane vesicle and its contents into the lysosome interior
Vesicle protein and lipid components – degraded by lysosomal hydrolases
Amino acids – permease transport across the lysosomal membrane into the cytosol for use in protein synthesis