Biol110-10-Lecture 11-Late Sec Pathway

Biol110-10-Lecture 11-Late Sec Pathway - The late secretory...

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Unformatted text preview: The late secretory pathway: A macrophage can ingest 25% of its volume every hour, and 100% of its plasma membrane every 30 min; thus endocytic and exocytic events must be highly regulated or coupled Constitutive Regulated Pinocytosis (drink) Phagocytosis (eat) Reading for next class: Alberts Chapter 15, pgs 879-912 & 921-931 Exocytosis: Protein sorting and exocytic vesicle formation in trans-Golgi membraneMost proteins and lipids are transported in vesicles and tubules “by default” from the trans-Golgi to the plasma membrane without need for sorting signals. Targeting signals or aggregation domains are needed for diversion from the default pathway to other pathways, such as early endosomes or regulated vesicles. Constitutive secretion is not ‘concentrative’ Clathrin-coated vesicles default Selective aggregation Un-regulated secretory tubules and regulated secretory vesiclesMast cells coordinately releasing histamine Connection to allergies… Exocytosis can be a localized response Maturation of secretory vesicles•  Secretory proteins aggregate at the site of exit and bud as an immature secretory vesicle •  Proteins are concentrated in secretory vesicles via selective retrieval of Golgi proteins. •  Protein processing by specific proteases occurs in the trans-Golgi membrane and late secretory vesicles •  Removal of ‘pro’ sequences triggers a final folding of the protein that activates it (i.e. hormones, neuropeptides). Why are these peptides made as larger proteins? Transport from the trans-Golgi network to lysosomes (vacuoles) occurs via early endosomes that mature (through sorting) to become late endosomes/lysosomes•  Lysosomes are full of acidic hydrolytic enzymes (~40 different types) •  Lysosomes arise from the maturation of late endosomes and have heterogeneous morphology •  Called vacuoles in plants and fungi (including yeast); there they can serve as storage place (e.g. seeds) •  Also contain lots of permeases for amino acids, nucleotides and sugar delivery to cytosol. •  Lysosomal permeases are protected from degradation by their high content of carbohydrates. •  A proton pump acidifies the lysosome interior and other compartments of the late secretory pathway Features of a lysosome Formation of lysosomes from late endosomes Acid phosphatase-stained lysosomes under TEM.. heterogeneous The lysosomes in plants/yeast are called vacuoles Mannose-6-phosphate sorting signals are added to lysosomal hydrolases in the cis-Golgi as tags for later sorting in the trans-Golgi The recognition site in hydrolase is encoded in the protein sequence; it is called a signal patch and forms in 3-D structure of proteins In the cis-Golgi In the trans-Golgi… Why? Lysosomal protein sorting at the trans-Golgi network (via clathrin-coated vesicles) and delivery to early endosomes M6P binds to a receptor in the trans-Golgi network, at pH ~6.6 The M6P receptor binds to clathrin adapters and is incorporated into vesicles The M6P receptor releases the M6P at pH 6, in early endosomes. The M6P receptor is captured in vesicles budding from late endosomes and is transported back to the trans-Golgi in retromer-coated vesicles Removal of targeting signal In I-cell disease the M6P transferase is missing and all soluble lysosomal hydrolases of fibroblasts are secreted to cell exterior (default pathway) due to inability to be packaged into vesicles Many other cellular pathways lead to the lysosomes1) Proteins unretrieved from early endosomes end up in late endosomes/lysosomes by default 2) Autophagosomes containing mitochondria, ER, etc. fuse with the lysosome 3) Phagosomes containing bacteria, dead cells, etc. fuse with the lysosomes 4) Multivesicular bodies containing down-regulated cell surface proteins fuse with lysosomes Everything that enters the lysosome will be digested to elementary units (AAs, nucleotides, etc) and exported to cytosol by specialized porins for re -use (recycling) trans-Golgi Autopaghy (cellular eating from interior sources/ cannibalism)Can remove whole organelles or large proteasome-resistant protein aggregates Can also remove viruses or bacteria that have invaded the cytoplasm Can engulf cytoplasmic proteins as food in case of starvation 3 2 ? 1 4 Example: in a liver cell, mitochondria have a lifespan of ~10 days; then they are degraded by autophagy. Phagocytosis (cellular eating from exterior sources)•  In mammals, macrophages, neutophils and dendritic cells (immune cells) specialize in phagocytosis. •  Phagocytosis is a triggered event, responding to binding of a particle to the plasma membrane e.g. (binding via antibodies coating a foreign particle). Antibodies are recognized by a receptor which signals formation of the phagosome. Dead cells are recognized by damaged phospholipids in the plasma membrane. •  Phagocytosis is accomplished by the restructuring of the actin cytoskeleton. •  Phagosomes delivers their contents directly to lysosomes Neutrophil eating a dividing bacteria Phagocytosis by a macrophage-SEM pseudopods Our macrophages phagocytose 10 billion red blood cells in our body every day…they also clear apoptotic cells Endocytosis and clathrin coated vesicles- These honeycomb structures are clathrin coated pits The dynamin GTPase- Actin and cofactors can propel the vesicles A large homotetrameric GTPase protein. Self-assembles into supramolecular structures, such as rings and spirals. Self-assembly stimulates dynamin’s GTPase activity. Important in all clathrin-mediated vesicle budding Severs nascent clathrin-coated vesicles from membranes, sometimes with the help from actin Shibire is a ts mutant in dynamin, It makes flies tremble at high temp Why dynamin? Because distance too large to push membranes together…? squeeze push Sorting signals for capture of PM transmembrane proteins into clathrin coated vesicles- GPI-anchored proteins are sorted into lipid rafts (for inclusion into caveolae) by their lipid groups Sorting signals are recognized by clathrin and/or its many adaptorsAt the amino terminus of each CHC there is globular domain known as the terminal domain (TD), which serves as a binding site for many adaptor proteins, as indicated in the figure. All of these proteins are part of coats associated with cytosolic faces of membranes. The sorting signals proposed to interact with each of these proteins are indicated. Atherosclerosis/familial hypercholesterolemia and receptor-mediated endocytosis Coated pits serve to trap and concentrate extracellular proteins in preparation for endocytosis Cellular uptake of LDL (the particle in blood that carries cholesterol) supplies cholesterol to its membranes Defect in uptake of LDL causes a build up of cholesterol in blood leading to atherosclerosis A mutation in the cytoplasmic domain of LDL-receptor (the Y residue) is the cause of familial hypercholoesterolemia Nobel Prize in Medicine or Physiology- Brown and Goldstein 1983 Receptor down-regulation via ubiquitination and multivesicular bodiesFast inactivation of cell surface signaling (via the cytoplasmic domain of receptor) by ‘sequestering’ the domain away from cytosol. Example EGF receptor. Such membrane proteins are tagged and sorted by the addition of ubiquitin Possible fates of proteins taken up via endocytosis (recycling, degradation)Four general pathways Transferrin and transferrin receptors are recycled after endocytosis LDL receptors are recycled after endocytosis; LDL is degraded Endocytosis via caveolae•  Caveolae are shaped by a protein called caveolin, a multipasss membrane protein •  Caveolae originate at lipid rafts which contain glycosphingolipids and cholesterol •  Materials are sorted into caveolae via their lipid composition, which in turn shapes the membrane into a vesicle without the need for protein coats •  Proteins with long-enough transmembrane domains partition into these rafts (sorting) •  Lectins may help stabilize the raft; internal scaffold •  These vesicles can be targeted to endosomes, ER or the plasma membrane at another side of the cell. Caveolae vesicles have no external coats Protein sorting in polarized cellsProtein asymmetry in polarized cells can be generated via selective targeting of vesicles derived from the trans-Golgi network Tight junctions are diffusional barriers for proteins and lipids (in the outer leaflet). Recycling endosomes and transcytosis: Transcytosis is the movement of proteins from one part of the plasma membrane to another, via recycling endosomes (e.g transport of antibodies across epithelial cells) Recycling endosome can function as storage sites for proteins (e.g. glucose transporter) Transport of antibodies (in mothers milk) from ‘gut to bloodstream’ across epithelial cells Storage of plasma membrane proteins in recycling endosomes Viral strategies for entry into cells and delivery of their genetic material into nuclei- HIV entry into cells requires membrane fusion via a fusogen: Viral strategies for evading immune detection: Mistargeting of newly synthesized MHC class I molecules by human cytomegalovirus proteinsExposure of MHC proteins in cell surface of immune cells is a key step in mounting an immune response. Viruses have developed strategies to prevent MHC presentation … US2 and US11 (viral proteins) bind MHC in the ER, and target it for dislocation via the Sec61 translocon Dislocated MHC is degraded in the cytoplasm by the proteasome M04 (viral protein) binds MHC and prevents its capture of immune peptides in the ER M06 binds MHC and redirects it to the lysosome via a M6P lysosomal targeting signal Biol110-09-Midterm Grade Curve and Totals 90-100 75-89 70-74 65-69 60-64 55-59 50-54 45-49 30-44 0-29 A+ A AB+ B BC+ C D F totals 33 A 47 B 26 C 38 D 15 F 45% midterm 45% final 10% quizes ~50% of total points (50/100 points final) is needed to pass regardless of curve 66% passed How to do better for the final? Attend class Read chapter before class, and review after class Study every day; total of more than 10 h /week (5 unit course) Do not fall behind Answer exam questions giving full information as in all keys posted Effort is necessary, but not sufficient; you need to make the grade All requests for re-grades need to be submitted in writing before Friday May 15 at 5 pm; no oral discussions about re-grades, especially during office hours. A specialized form of secretory vesicles (synaptic vesicles) in nerve cells- Intracellular location of phosphoinositides in the late secretory pathway: Adding specificity to trafficking events: ...
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