BIOL 3510 Lecture 15 Intracellular Transport PDF

BIOL 3510: Lecture 15 Intracellular transport II (Ch. 15) Vesicular transport Secretory pathways Endocytic pathways Objectives: ▪ Define vesicular transport, secretory pathway and endocytic pathway. ▪ Explain the process and role of each of the molecular components involved in...

BIOL 3510: Lecture 15 Intracellular transport II (Ch. 15) Vesicular transport Secretory pathways Endocytic pathways Objectives: ▪ Define vesicular transport, secretory pathway and endocytic pathway. ▪ Explain the process and role of each of the molecular components involved in vesicle budding, docking, and fusing. ▪ Recognize examples of how proteins are chemically modified in the ER and Golgi. Describe the purpose and outcomes of the unfolded protein response. ▪ Describe the processes and organelles involved in exocytosis. Compare/contrast regulated vs. constitutive secretion. ▪ Describe the processes and organelles involved in endocytosis. Compare/contrast phagocytosis vs. pinocytosis vs. receptor-mediated endocytosis vs. autophagy. Describe the example of cholesterol uptake by receptor-mediated endocytosis. ▪ List specialized properties and processes involved in the endosome and lysosome. Mechanisms of proteins transport Membrane-enclosed organelles import proteins by one of three mechanisms: hand 1. Gated transport: folded proteins enter though pores 2. Transmembrane transport: proteins enter via protein translocators shined 3. Vesicular transport: folded proteins move via transport vesicles that fuse with destination membranes gut L True or False: protein import into organelles generally requires energy.  true Vesicular transport ▪ Vesicular transport moves proteins and lipids between membrane compartments. ▪ Secretory pathway (red) - ER to Golgi to plasma membrane - ER to Golgi to endosome to lysosome ▪ Endocytic pathway (green): plasma membrane to lysosome ▪ Retrieval pathways (blue) bring membrane and selected proteins back to the original compartment.  Vesicle budding ▪ Vesicles that bud from membrane have a distinctive protein coat on the cytosolic surface. ▪ Clathrin molecules form basketlike cages that help shape A membranes into vesicles. The structure of a clathrin coat vesicles ▪ The major protein component of clathrin-coated vesicles is clathrin itself, which forms the outer layer of the coat. ▪ Each clathrin subunit consists of three large and three small polypeptide chains that together form a three-legged structure called a triskelion. ▪ Triskelions can spontaneously self-assemble into typical polyhedral cages.  Vesicle budding driven by the assembly of a protein coat Clathrin-coated vesicles formation: ▪ cargo receptors recognize transport signals on cargo molecules and bind ▪ adaptins mediate the connection between coat proteins and cargo receptors ▪ clathrin protein molecules form basketlike cages that help shape membranes into vesicles. ▪ dynamin (GTP-binding protein) pinches off the vesicle thenchased dissembles me D Different types of coated vesicles ▪ COP = ‘coat protein’ ▪ COPII: mediates anterograde transport from the ER to the Golgi. ▪ COPI: involved in retrograde transport from the Golgi back to the ER. t ▪ Clathrin: participates in endocytosis at the plasma membrane and transport between the Golgi and endosomes. How do vesicles move Anterograde orproto ▪ Kinesins are responsible for moving cargo from the cell body toward the cell periphery (anterograde f transport). mm ▪ Dynein facilitates moving cargo back toward the cell body (retrograde transport). Q: which motor protein is used to move vesicles from ER to Golgi apparatus? Kinesins Vesicle reaches target…Recognition…Vesicle docking…Unload Cargo Protein-protein interactions mediate fusion of transport vesicles with appropriate membranes. vesicle membrane tethering tethering Rab protein docking v-SNARE t-SNARE fusion v-SNARE t-SNARE Tethering protein = Rab effector Rab proteins play a central part in the specificity of vesicle transport  Vesicle fusion ▪ Vesicle fusion is mediated by SNARE proteins. ▪ The v-SNAREs and t-SNAREs wrap around each other tightly, pulling the two lipid bilayers into close proximity. ▪ FUSION = delivers cargo AND membrane iClicker Question True/False. Each of these structures is correctly matched for receptor-mediated endocytosis with their functions. Cargo = Molecules packaged into vesicle for transport Receptor = Captures the correct cargo molecules Clathrin = Shapes the forming vesicle i Adaptin = Mediates contact between the receptor and another component A. True B. False mywor Secretory Pathways - Exocytosis ▪ Protein modification in ER, exit from the ER ▪ Modification in Golgi ▪ Exocytosis release processes ▪ Only those proteins that achieve the correct configuration make it to the cell surface Q: which coat protein is involved in the early stages of exocytosis? COPI  Most proteins are modified in the ER Most proteins are covalently modified in the ER by enzymes: 1. Disulfide bond formation - protein stabilization 2. Glycosylation – addition of oligosaccharide side chains (next slide) - protein protection from degradation - keep the protein in the ER for proper folding - act as transport signal for packaging into vesicle - cell-cell recognition (surface carb layer glycocalyx)  Protein modification in the ER If aprotein is misfolded it Preformed oligosaccharides are transferred to asparagines by oligosaccharyl transferases. cannot leave the ER anti ▪ Amino acid sequence on protein necessary and sufficient for N-linked glycosylation: Asn-X-Ser/Thr ▪ Each oligosaccharide chain is transferred as an intact unit to the Asn from a lipid called dolichol. ▪ Oligosaccharides are further modified in the ER and Golgi apparatus.  Exit from the ER ▪ Exit from the ER is controlled to ensure protein quality. ▪ Proteins don’t leave if they: - have an ER retention signal (KDEL at C-terminus) - are misfolded → retained by chaperones - are incorrectly assembled into multimeric proteins → retained by chaperones  Unfolded protein response (UPR) Iffoldingarrangement ▪ Triggers increase in ER and folding machinery (chaperones) is taking too long ▪ Slows down ER protein synthesis to allow folding machinery to catch upwill make Cell more chaperones so thatthey go to ER and properly Shorpe Proteins Response could be detrimental Proteinsmay betrappedin ER  The Golgi network ▪ cis Golgi network – faces the ER - buds vesicles that return to the ER or move through the Golgi apparatus ▪ trans Golgi network – faces the plasma membrane - buds vesicle headed to the plasma True or False: vesicles from ER fuse membrane or other compartments with trans Golgi network. False  Further modification & sorting in the Golgi ▪ Individual stacks have different assortments of enzymes, allowing for progressive Ñ processing of cargo proteins as they travel to the trans Golgi face. ▪ Proteins exiting from the trans Golgi network move onward and are sorted according to their next destination. Man: mannose; GlcNAc: N-acetylglucosamine; Gal: galactose; NANA: N-acetylneuraminic acid (sialic acid)  Exocytosis Proteins (and lipids) move to the plasma membrane via 2 different pathways: gammranpacksides 1) Constitutive secretion - default, no signal sequence - continuous operation 2) Regulated secretion - only in some cells - proteins aggregate - products stored in secretory vesicles until signal is received Exocytosis An example of regulated secretion: ▪ Increase in glucose level in the blood causes the release of insulin from pancreatic β cells. ▪ Involves glucose metabolism, ATP-sensitive K+ channels, voltage-gated Ca2+ channels, SNARE proteins. True or False: increased ATP level The activates K+ channels in this process. process includes depolarization hyperpolarization  by iClicker Question What is the primary role of the Golgi apparatus in the vesicular transport pathway following cargo arrival from the ER? A. Protein synthesis. B. Protein sorting and modification. C. Protein degradation. D. DNA and RNA synthesis.  Endocytosis Endocytosis is the uptake of material into a cell by membrane invagination and internalization of the resulting vesicle. 1) Phagocytosis is the uptake of particles into large vesicles (phagosomes) - occurs in phagocytic cells (dendritic cells, macrophages, and neutrophils) - Phagosomes fuse with lysosomes  Endocytosis 2) Pinocytosis is the uptake of fluids and molecules in small vesicles. - Occurs via clathrin-coated pits and vesicles - Balanced with the rate of exocytosis - Vesicles fuse with endosomes The True or False: pinocytosis is generally a non-specific process.  1 sept Endocytosis 3) Receptor-mediated endocytosis is the selective endocytosis of specific macromolecules. LDL – low density lipoproteins (cholesterol + proteins) Viruses can enter cells via receptor-mediated endocytosis A ▪ These vesicles will fuse with lysosomes, sometimes resulting in the release of the viral genome into the cytoplasm and its subsequent replication. Endosome = sorting compartment receptors for the tolysosome back orto goanotherpart or get of cell 1. Recycling – receptors return to the same plasma membrane 14kg 2. Degradation – receptors/cargo and contents of the lumen move to lysosomes 3. Transcytosis – receptors/cargo move to a new plasma membrane area True or False: in receptor-mediated endocytosis, A the fate of receptor proteins following their endocytosis depends on the type of receptor. Epithelial cell true Lysosome = site of intracellular digestion ▪ Lysosomes digest extracellular materials and old organelles. ▪ Acidic: contains H+ pump ▪ Transporters that export metabolites. ▪ Membrane proteins are glycosylated for protection. ▪ Signal sequence: mannose-6- phosphate (M6P) sorted at trans Golgi network. Lysosome = site of intracellular digestion Pathway to lysosomes: Autophagy: enclosure of an old organelle in a double membrane creating an autophagosome iClicker Question Which of the following terms could be defined as uptake of large particles by certain cells, primarily immune cells like macrophages and neutrophils? A. Phagocytosis B. Exocytosis C. Autophagy D. Pinocytosis