PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Intracellular Transport 1Intracellular TransportChapter 15You should review functions of different organellesWe already discussed evolution; reviewFocus will be on:1) mechanisms of protein targeting and transport to organelles2) vesicle formation and targeting to organelles and cell surfaceQuestions in this chapter you should be able to answer:Chapter 15: 1 - 8, 9 (all but B),10, 12 - 16, 18,19, 21, 22Cell CompartmentsIntracellular Transport 2How did internal membranes arise?Cytosolic membranesOrganelle membranesIntracellular Transport 3Proteins are synthesized in the cytosol…How do they get into appropriate organelles?Three mechanisms:1) nuclear pores (nucleus)2) protein translocators (ER, mitochondria, chloroplasts, peroxisomes)3) vesicular transport(golgi, lysosomes, cell surface)Intracellular Transport 4How are proteins ‘targeted’ to appropriate membrane?Signal sequences-- N-terminus-- protein surfaceIntracellular Transport 5How do proteins pass into chloroplasts and mitochondria?‘Post-translational’ transmembrane transportN-terminal signal sequence cleavedPost-translational transportIntracellular Transport 6How do we know that signal peptides are necessary?In vitro translate mRNA for a mitochondrial protein-- radiolabeled (e.g., with 35S)-- assume 55Kd with signal peptide; 53 Kd without signal peptide-- incubate with isolated mitochondria; run through density gradient centrifugationWhy does each type of protein occur in the fraction in which it is found?If SDS-PAGE is then performed for the proteins from the two experiments, what would be the molecular weight of the labeled proteins?Intracellular Transport 7How do proteins pass into ER?‘Co-translational’ transmembrane transportSignal recognition particle & receptorProtein translocator complexChaperone proteins aid in folding“rough ER”Intracellular Transport 8How are transmembrane proteins inserted in the membrane?Single-pass transmembrane proteins-- have a stop-transfer sequenceMulti-pass proteins will have multiple ‘start-transfer’ and ‘stop-transfer’ sequencesCo-translational transport overviewIntracellular Transport 9What happens to proteins after translation?Covalent modificationse.g. glycosylationacetylationmethylationprosthetic groupschemical modificationsetc.Preassembly on dolichol-PIntracellular Transport 10How do proteins pass through nuclear pores?Nuclear localization sequences-- a ‘patch’ on protein surfacePores & nuclear transport receptors-- requires GTP hydrolysisQuestionOften nuclear localization signals are not removed from proteins after import into the nucleus. Why? When might they need to be reused?ImportinNucleoporinIntracellular Transport 11What is the most recent model for nuclear import? (see Fig 15-10)Importins (kapβ) nuclear transport receptors that bind Nuclear Localization SequencesNucleoporins form a gel-like barrier -- have extended phe-gly (FG) repeatsImportins cause contraction of nucleoporinsLim, et al. 2007. Nanomechanical Basis of Selective Gating by the Nuclear Pore Complex. Science 318: 640 - 643RanGTP allows dissociation & re-export of importin-- GTP  GDP hydrolysis powers export to cytosolIntracellular Transport 12What is the path of vesicular transport?The endomembrane transport pathwayWhat are the 3 steps of vesicular transport?1) vesicle budding2) targeting3) fusionGFP-labeled vesicular stomatitis virus membrane protein from ER to the cell membrane in cultured cells Two-way transport of GFP-labeled protein between Golgi and axonal membraneVSV transportAxonal transportIntracellular Transport 13What is the mechanism of vesicle formation?Sorting and packaging are coupled= receptor mediated (endocytosis)Question 15-5P 506Roles of COPI, COPII & ClathrinClathrin Receptor-mediatedIntracellular Transport 14What are the mechanisms ofvesicle targeting and fusion?Targetting: Rab proteins & Rab-effectors Fusion & targetting: t- and v-SNARESEnergy dependent GTPases Question 15-12 page 529Intracellular Transport 15What is the structure and function of the Golgi apparatus?Protein processing & sortingIntracellular Transport 16How does transport occur through the Golgi apparatus?Why does retrograde transport occur?KDEL & ER resident proteinsretrogradeanterogradeER sideIntracellular Transport 17What are Regulated and Constitutive secretion?Examples:Insulin secretionMucusneurotransmittersRegulated secretion & Sea Urchin egg fertilizationIntracellular Transport 18Adapted from Question 15-8Fe is transported through blood on protein called transferrin, which can bind to the transferrin receptor on the surface of cells.Consider these observations:1. Fe only binds to transferrin at neutral pH2. Transferrin/Fe only binds to its receptor at neutral pH3. ‘Empty’ transferrin only binds to its receptor at acidic pH4. The transferrin receptor continually cycles from the cell surface to endosomes, and then back to cell surface.Describe processes & mechanisms that would answer these question:A.After being endocytosed into the cell, transferrin and its receptor will move into which compartment? B.What happens within this compartment to the FE, transferrin and receptor?C.Why aren’t the proteins transported to lysosomes?D.What are the advantages of this system?Intracellular Transport 19What are the functions of and pathways to the lysosome?Vesicular transport from the cell membrane-- endocytosis, phagocytosis vs pinocytosisHow are hydrolases targeted to lysosome? --