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Alberts • Johnson • Lewis • Raff • Roberts • Walter Molecular Biology of the Cell Fifth Edition Chapter 13 Intracellular Vesicular Traffic Copyright © Garland Science 2008 The cell continually adjusts the composition of its plasma membrane Figure 13-1 Molecular Biology of the Cell (© Garland Science 2008) Transport vesicles bud from one membrane and fuse with another, carrying a cargo (soluble molecules in it) Figure 13-2 Molecular Biology of the Cell (© Garland Science 2008) The “road-map” of the biosynthetic secretory and endocytic pathway Figure 13-3 Molecular Biology of the Cell (© Garland Science 2008) Figure 13-3b Molecular Biology of the Cell (© Garland Science 2008) Vesicular transport • Mediates continuous exchange of components between chemically distinct membrane-enclosed compartments. • molecular markers displayed on the cytosolic surface of the membrane serve as guidance for traffic, to ensure that vesicles fuse only with the correct compartment. • A specific combination of markers gives each compartment its molecular address • Cells segregate (accumulate in one space) proteins into separate membrane domains by assembling a special protein coat on the membrane’s cytosolic surface. Coated vesicles • Coats are needed for budding • Before fusion happen the coats are removed from the vesicles • The function of the coats is to: - Concentrate specific membrane proteins in a specific patch, which then makes a vesicle - Coat molds (shapes) the forming vesicle Coat proteins assemble into a basket like curved lattice, and so shapes the vesicle. • Same types of coats make similar shaped / sized vesicles Three different types of coats cover transport vesicles Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008) Each coat is used for a different transport step Figure 13-5 Molecular Biology of the Cell (© Garland Science 2008) Clathrin coated pits Figure 13-6 Molecular Biology of the Cell (© Garland Science 2008) Clathrin protein is the major component f clathrin-coated vesicles Composed of 3 large and three small polypeptides, together form the triskelion Figure 13-7a, b Molecular Biology of the Cell (© Garland Science 2008) The clathrin triskelion determines the geometry of the clathrin cage Figure 13-7c, d Molecular Biology of the Cell (© Garland Science 2008) Adaptor proteins are a major component in clathrin-coated vesicles, they form a second layer of the coat, they capture cargo receptors The sequencial assembly of adaptor complexes and clathrin on the cytosolic side of the membrane generate forces that results in the formation of a clathrin-coated vesicle Figure 13-8 Molecular Biology of the Cell (© Garland Science 2008) Not all coats form basket-like structures: retromer assemble on endosomes to Golgi Figure 13-9 Molecular Biology of the Cell (© Garland Science 2008) RETROMER ASSEMBLY Retromer assembles when: 1. It can bind to the cytoplasmic side of the cargo receptors 2. It can interact directly with a curved phospholipid bilayer 3. It can bind to a specific phospholipid: phosphorylated phosphatydilinositol, which act as an endosomal maker. PHOSPHOINOSITIDES MARK ORGANELLES AND MEMBRANE DOMAINS • Phosphoinositides can undergo rapid cycles of phosphorylation and dephosphorylation at te 3’, 4’ and 5’ end to produce various PIPs. • Different organelles in the endocytic and secretory pathway have distinct sets of PI and PIP Kinases and phosphatases. Figure 13-10 Molecular Biology of the Cell (© Garland Science 2008) PIP-BINDING PROTEINS HELP REGULATE VESICLE FORMATION AND OTHER STEPS IN MEMBRANE TRANSPORT Figure 13-11 Molecular Biology of the Cell (© Garland Science 2008) Cytoplasmic proteins regulate the pinching-off and un-coating of coated vesicles • Dynamin assemble as a ring around the neck of each bud, using GTP energy it works to help seal off the forming vesicle. Dynamin binds to PI(4,5)P2 which tethers the protein on the membrane. • After vesicle forms, clathrin falls off with the help of an Hsp70 ATPase; a PIP phosphatase that is in the vesicles depletes PI(4,5)P2 Figure 13-12a Molecular Biology of the Cell (© Garland Science 2008) Cytoplasmic proteins regulate the pinching-off and un-coating of coated vesicles- role of dynamin Figure 13-12b Molecular Biology of the Cell (© Garland Science 2008) Monomeric GTPases control coat assembly To balance vesicular traffic to / from compartment, coat proteins must assemble only when and where they are needed: • PIPs play a major role in regulating the assembly of clathrin coats on the PM and golgi, there are in addion: • Coat-recruitment monomeric GTPases: control the assembly on endosomes and the COPI and COPII coats on golgi and ER: • These include Arf proteins (COPI and clathrin at golgi) • Sar1 (COPII on ER) Usually these are found in the cytosol at high concentrations, in an inactive GDP-state. Monomeric GTPases control coat assembly: Sar1 activation Figure 13-13a Molecular Biology of the Cell (© Garland Science 2008) Active Sar1 GTP now recruits coat (COPII) protein subunits to initiate budding Figure 13-13b Molecular Biology of the Cell (© Garland Science 2008) COPII protein components Figure 13-13d Molecular Biology of the Cell (© Garland Science 2008) Sec13/31 assemble into a cage Figure 13-13c Molecular Biology of the Cell (© Garland Science 2008) NOT ALL TRANSPORT VESICLES ARE SPHERICAL • Clathrin coats have to produce considerable force to bend the plasma membrane, especially at the neck of the bud where Dynamin is required for pinching of he vesicle. • Budding occurs preferentially at regions where the membrane are already curved, such as the rims of the Golgi cisternae. • Vesicles are dynamic and of different shapes and sizes: long tubules leave the trans Golgi network, vesicle pinch off from the tubules. RAB PROTEINS GUIDE VESICLE TARGETING •Rabs are monomeric GTPases: they can function on transport vesicles, on target membranes or both. • They cycle between the cytoplasm and the membrane (regulated by GAPs and GTPases). • Rab GDI (Rab-GDP dissociation inhibitor) binds to GDP-bound state to keep them soluble inactive in the cytoplasm. • In their GTP state they bind to effectors called: Rab effectors that facilitate vesicle transport, membrane tethering and fusion. RAB PROTEINS GUIDE VESICLE TARGETING Table 13-1 Molecular Biology of the Cell (© Garland Science 2008) RAB PROTEINS ROLE IN TETHERING DOCKING AND FUSION Figure 13-14 Molecular Biology of the Cell (© Garland Science 2008) SNAREs MEDIATE MEMBRANE FUSION Figure 13-16 Molecular Biology of the Cell (© Garland Science 2008) SNAREs MEDIATE MEMBRANE FUSION • After tethering, the vesicle fuses to the target membrane: • Specialized fusion proteins overcome energy barrier to catalyze membrane fusion: • SNARE proteins do this and also participate in specificity (35 different SNAREs) • SNAREs exist as complementary sets: v-SNAREs (a single protein) on vesicles and t-SNAREs (two or three proteins) on target membranes. A model of how SNARE proteins mediate membrane fusion Figure 13-17 Molecular Biology of the Cell (© Garland Science 2008) SNAREs must be disassembled before they can function in each fusion events: NSF ATPase does that Figure 13-18 Molecular Biology of the Cell (© Garland Science 2008) Skip Viral fusion paragraph 764-766 Page 766 Molecular Biology of the Cell (© Garland Science 2008) The recruitment of cargo molecules into ER transport vesicles (COPII) ER exit sites: where vesicles bud Figure 13-20 Molecular Biology of the Cell (© Garland Science 2008) To exit the ER proteins must be properly folded: chaperones help in this folding. Figure 13-21 Molecular Biology of the Cell (© Garland Science 2008) Homotypic fusion: fusion of membranes from the same compartment: requires matching SNAREs Figure 13-22 Molecular Biology of the Cell (© Garland Science 2008) Structures that form when ER derived vesicles fuse together: Vesicular-tubular clusters, making a new compartment. Figure 13-23a Molecular Biology of the Cell (© Garland Science 2008) Structures that form when ER derived vesicles fuse together: Vesicular-tubular clusters, making a new compartment; short lived, move along micotubules. Retrieval, retrograde transport from the vesicular tubular clusters happens by COPI vesicles. Figure 13-23b Molecular Biology of the Cell (© Garland Science 2008) The retrieval pathway to the ER uses sorting signals: KDEL is a resident sequence Figure 13-24a Molecular Biology of the Cell (© Garland Science 2008) KDEL receptor is needed, it has high affinity to cargo in VTC and low affinity in the ER Figure 13-24b Molecular Biology of the Cell (© Garland Science 2008) The Golgi apparatus consists of an ordered series of compartments: 4-6 cisternae in the Golgi Figure 13-25a Molecular Biology of the Cell (© Garland Science 2008) The Golgi is localized near the nucleus, held by microtubules Figure 13-25b Molecular Biology of the Cell (© Garland Science 2008) Each Golgi has two distinct faces: cis face (entry) and trans face (exit): CGN and TGN Figure 13-25c Molecular Biology of the Cell (© Garland Science 2008) Figure 13-26a Molecular Biology of the Cell (© Garland Science 2008) Different enzymes localize to different Golgi areas Figure 13-27 Molecular Biology of the Cell (© Garland Science 2008) Different enzymes localize to different Golgi areas Figure 13-28 Molecular Biology of the Cell (© Garland Science 2008) The Golgi is prominent in cells that are specialized for secretion: goblet cells secrete mucus in the intestinal epithelium Figure 13-29 Molecular Biology of the Cell (© Garland Science 2008) DIFFERENT ENZYMES LOCALIZE TO DIFFERENT GOLGI AREAS Resident Golgi proteins are all membrane bound, So the ER and the Golgi are organized in different ways. OLIGOSACCHARIDES ARE PROCESSED IN THE GOLGI APPARATUS: 2 classes of N-linked oligosaccharides Figure 13-30 Molecular Biology of the Cell (© Garland Science 2008) Ordered pathway of sugar modifications in the Golgi Figure 13-31 Molecular Biology of the Cell (© Garland Science 2008) N- and O- linked glycosylation Figure 13-32 Molecular Biology of the Cell (© Garland Science 2008) DIFFERENT ENZYMES LOCALIZE TO DIFFERENT GOLGI AREAS • N-linked glycosylation promotes protein folding: Makes folding intermediates more soluble, therfore preventing their aggregation. the sequential modifications of the N-linked oligosaccharides establish a code that helps chaperones in binding. • Makes a protein more resistant to digestion • Protection against pathogens • cell – cell recognition : development • Signaling Transport through the Golgi may occur by Vesicular transport or by Cisternal maturation Figure 13-35 Molecular Biology of the Cell (© Garland Science 2008) Figure 13-35a Molecular Biology of the Cell (© Garland Science 2008) Figure 13-35b Molecular Biology of the Cell (© Garland Science 2008) Page 779 Molecular Biology of the Cell (© Garland Science 2008) Lysosomes contain soluble hydrolytic enzymes • Lysosomes membrane are highly glycosylated, well protected from the lysosomal proteases • Enzymes used for digestion of macromolecules Figure 13-36 Molecular Biology of the Cell (© Garland Science 2008) LYSOSOMES ARE HETEROGENEOUS IN SHAPE AND SIZE Figure 13-37 Molecular Biology of the Cell (© Garland Science 2008) MODEL FOR LYSOSOME MATURATION No real distinction between lysosomes and late endosomes, they are at different stages of the maturation cycle. Figure 13-38 Molecular Biology of the Cell (© Garland Science 2008) Skip p781 section “plant…” MULTIPLE PATHWAYS DELIVER MATERIALS TO LYSOSOMES From ER golgi lysosomes Other pathways also include: • From endocytosed material early endosomes late endosomes • Autophagy: in liver cells for ex. the mitochondria lives 10 days Autophagy process is highly regulated and selected cell components can be marked for lysosomal destruction: form autophagosomes; can remove large organelles or protein complexes. MODEL OF AUTOPHAGY 4 steps in autophagy: 1. Nucleation and extension, englufs part of cytoplasm 2. Closure of autophagosome 3. Fusion with lysosomes 4. Digestion of the inner membrane of the autophagosome Figure 13-41 Molecular Biology of the Cell (© Garland Science 2008) SUMMARY OF THREE PATHWAYS TO DELIVER MATERIAL TO LYSOSOMES Figure 13-42 Molecular Biology of the Cell (© Garland Science 2008) TRANSPORT OF PROTEINS TO LYSOSOMES • Proteins are co-translationally transported into the rough ER, then go to the Golgi and TGN • In the TGN proteins carry a marker mannose 6-phosphate (M6P) groups added to the N-linked oligosaccharides • M6P receptors in the TGN recognize these , cclathrin coat assembles and the vesicles are delivered to the early endosomes. M6P receptor binds to M6P at high pH and release it at low pH. Figure 13-43 Molecular Biology of the Cell (© Garland Science 2008) TRANSPORT OF NEWLY SYNTHESIZED LYSOSOMAL HYDROLASES TO LYSOSOMES Note: retromer is needed for recycling the M6P receptor to the golgi Figure 13-44 Molecular Biology of the Cell (© Garland Science 2008) A signal patch in the hydrolase polypeptide chain provides the cue for M6P addition • M6P are added only to the appropriate glycoprotein in the golgi apparatus • a Signal patch is the signal to add the M6P • Two enzymes work sequencially: GlcNAc phosphotransferase in cis golgi adds GlcNAc-phosphate; a second enzyme in the trans golgi cleaves the GlcNAc leaving behind the M6P marker. Figure 13-45 Molecular Biology of the Cell (© Garland Science 2008) DISEASES RELATED TO LYSOSOMES Genetic defects that affect the lysosomal hydrolases cause lysosomal storage diseases: results in an accumulation of undigested substrates in lysosomes , with sever pathological consequences, in nervous system. In Inclusion-cell disease, almost all of the hydrolytic enzymes are missing from the lysosomes of fibroblasts, forming large inclusions, the hydrolases fail to sort properly so they are secreted to the blood, because of a defective GlcNAc phosphotransferase. SOME LYSOSOMES DO EXOCYTOSIS • In skin cells, the pigment making cells called melancytes store their piment in lysosome compartments called malanosomes. • The melanosomes fuse with the membrane to empty the pigments which are then taken up by kratinocytes, leading to bormal skin pigmentation. • In some genetic disorders defects of melanosome exocytosis leads to hypopigmentation = albinism Page 787 Molecular Biology of the Cell (© Garland Science 2008) Phagocytosis : cell uses a large endocytic vesicle: phagosome to ingest large particles • Professional phagocytes: Macrophages and neutrophils, defend against infections • the phagosome fuse with lysosomes inside the cell and ingested material is degraded. Figure 13-46 Molecular Biology of the Cell (© Garland Science 2008) • Phagocytosis is triggered process: it requires activation of receptors that transmit signals. The phagosome fuse with lysosomes inside the cell and ingested material is degraded. trigger is an antibody that binds to the infectious microorganisms forming a coat that recognized by macrophages and neutrophils. • Pinocytosis is constitutive: occurs continuously, regardless of the cell needs. Phagocytosis: a neutrophil reshaping a plasma membrane, forming a pseudopod Figure 13-47a Molecular Biology of the Cell (© Garland Science 2008) Pseudopod formation requires reshaping of actin and PI3 kinase required for closure of the phagosome. Figure 13-47b Molecular Biology of the Cell (© Garland Science 2008) Pinocytosis is continually occuring (cell size remains the same. So endocytosios = exocytosis) Formation of clathrin coated pit for processes of endocytosis Figure 13-48 Molecular Biology of the Cell (© Garland Science 2008) Not all pinocytosis vesicles are clathrin coated Caveolae is needed Figure 13-49 Molecular Biology of the Cell (© Garland Science 2008) CAVEOLAE • Form membrane microdomains – lipid rafts, rich in cholesterol and glycosphingolipids and GPI-anchored membrane proteins • Major protein in caveolae is caveolin – integral membrane protein • Caveolae vesicles use Dynamin to pinch off • Caveolins do not dissociate from formed vesicles, so they are delivered to the target compartments. RECEPTOR MEDIATED ENDOCYTOSIS • Takes up specific macromolecules from the extracellular fluid. • Macromolecules bind to specific receptors and are taken up by clathrin coated vesicles. • For example, cholesterol is taken up by receptor mediated endocytosis ; if this is blocked then cholesterol will accumulate in blood vessels causing atherosclerosis, plaques deposits that cause strokes and heart attacks. • Most Cholesterol is carried in blood as lipid-protein particle: low density lipoprotein (LDL), when a cell needs cholesterol for membrane synthesis it makes the receptor for LDL. LDL particle Figure 13-50 Molecular Biology of the Cell (© Garland Science 2008) RECEPTOR MEDIATED ENDOCYTOSIS OF CHOLESTEROL: vesicles are targeted to early endosomes, LDL is released lysosomes, here it gets hydrolyzed to make free cholesterol cell uses it to make membranes Figure 13-51 Molecular Biology of the Cell (© Garland Science 2008) Possible fates for transmembrane receptor proteins that have been endocytosed Figure 13-52 Molecular Biology of the Cell (© Garland Science 2008) Receptor mediated endocytosis of LDL: EE as a sorting station Figure 13-53 Molecular Biology of the Cell (© Garland Science 2008) RECEPTOR MEDIATED ENDOCYTOSIS OF TRANSFERRIN • Transferrin is a protein that binds to iron • It is taken up by receptor-mediated endocytosis • Both receptor and transferrin bound to iron go to EE • Iron is released, and the receptor with transferrin are recycled back to the PM. • At the neutral pH of the extracellular fluid, the empty transferrin is released. • Clathrin-dependent receptor mediated endocytosis is highly regulated: ubiquitin tagging, by mono-ub or multi-ub is needed. (remember poly-ubiquitination – a long chain- targets for degradation in proteosaome, it is different from mono (1 ub) or poly (many mono-) that targets for receptor mediated endocytosis. Figure 13-54 Molecular Biology of the Cell (© Garland Science 2008) Details of endocytic pathway from PM to lysosomes Figure 13-56 Molecular Biology of the Cell (© Garland Science 2008) Figure 13-57 Molecular Biology of the Cell (© Garland Science 2008) Skip any sections not included in this slide presentation Receptors can be “stored” in recycling endosomes to be used when needed (this is faster than having to express from the gene level) Figure 13-61 Molecular Biology of the Cell (© Garland Science 2008) Page 799 Molecular Biology of the Cell (© Garland Science 2008) TWO PATHWAYS FOR EXOCYTOSIS : CONSTITUTIVE AND REGULATED Figure 13-63 Molecular Biology of the Cell (© Garland Science 2008) The three best understood pathways for protein sorting in the trans Golgi Network Figure 13-64 Molecular Biology of the Cell (© Garland Science 2008) Exocytosis of secretory vesicles Figure 13-66a Molecular Biology of the Cell (© Garland Science 2008) Figure 13-66b Molecular Biology of the Cell (© Garland Science 2008) Secretory vesicles wait near the PM until signaled to release their contents (such as Calcium entry into cells) Figure 13-68 Molecular Biology of the Cell (© Garland Science 2008) Examples of exocytosis leading to membrane enlargement Figure 13-70 Molecular Biology of the Cell (© Garland Science 2008) Exocytosis has to be targeted to the correct membrane domain Figure 13-71 Molecular Biology of the Cell (© Garland Science 2008) Two ways of sorting PM proteins in a polarized epithelial cell Transcytosis Figure 13-72 Molecular Biology of the Cell (© Garland Science 2008) The formation of synaptic vesicles Figure 13-73 Molecular Biology of the Cell (© Garland Science 2008)