Lecture 2911/14/12holes between spokes allows movement of proteins that are smallsmall: <20,000 Ddiameter of gaps: 9 nmproteins can diffuse through these pore complexesproteins that are larger, like enzymes, are too large to diffuse so are brought into nucleus with a different mechanismoocytes were used to test permeability of nucleus. Using a needle to inject something into the cytoplasm.Injected gold particles, manufactured with different diameters.it was noted that particles of less than 9 nm would make it into the nucleus.gold particles were linked with nuclear localization signalsif with nuclear localization signals, molecules that are much larger are able to make it into the cellmRNA, ribosomes must also go through these pore complexesProtein in cytoplasm moving to nucleus:polymerase, helicase, histones, transcription factorswhen cargo proteins are available, bind to importins (alpha, beta subunits)nuclear localization signals bind to alpha subunitbeta will function to recognize specific pore complexes in order to move cargo into nucleusonce in nucleus, importin and cargo complex are competed for willRAN-GTP has affinity for importin. Competes with the cargo. Removes importin from cargoenzyme in cytoplasm: GTPase activation proteininteracts with RAN and causes GTP to become hydrolyzedRAN-GDP falls off and importin is able to be recyclednot clear how RAN returns to nucleus – may diffuse through pore complexonce back in nucleus, RAN will become recharged with GTP and RAN is now activated and can start againGEF: GTP exchange factorFrom Nucleus to CytoplasmNuclear Export Signalwill bind to exportin.Exportin binds to RAN-GTP.Compelx will move along pore complexes to leave. GTP is hydrolyzed.Cargo and exportin separate.RAN and exportin are recycledRAN GTP has affinity for importin, upon hydrolyzing affinity is lostMovement of Nucleic Acids:mRNA made in nucleus (3 prime poly A tail and 5’ CAP structure)as it is matured in nucleus, specific porteins will bind to mRNA and help move through pore complexproteins move along fibersno affirmative data now for a single methodPeroxisomal Proteins:oxidative enzymes, envolved it break down of fatty acidsassembled post translationEND OF EXAM 3Cellular Respiration: BioenergeticsGlycolysisGlucose will be broken down into 2 subunits: 2 pyruvate moleculesCitric acid cycle: pyruvate goes through cycle, CO2 comes outtake a liver cell where these enzymes are present, proteins made are able to go back to the original thing. Pyruvate to alanine. Alanine to pyruvate*no structures, enzymesMitochondrial events of citric acid cycle:aspartate, amino acids, purine, pyrimidines: convert amino acids to oxaloacetatesmitochondriapyruvate goes into mitochondria. CO2 is releasedremeber: what NAD and NADH do?be able to reconnize structure vs FAD and CoAwith another NAD reduced, 2 other CO2’s reduced with each NAD reduced.GTP reducedFAD -> FADHNAD NADHpyruvate has been changed to CO2end up with 6 CO22 ATP2 GTP1- NADH + 10 H2 FAD --> 2 FADH2energy comes from energy residing in produced moleculeUse energy just made ot make ATP in mitochondriaas NADH oxidized inside mitochondria, H+ move across into inner membrane space. How Is electron gradient made to oxidize NADH?Energy of oxidation glucose is now stored as the protons in the IMMWill reduce ATPOxidation phosphyrlationHow to tell proton moved across IMM?Red and Green lighted picture:green around nucleus isstrucutres are mainitained with interacting with cytoskeletonpermeability: IMM- very permeableouter membrane: macromolecules are able to move through IMMpH of inner membrane space is similar to that of cytoplasmHow do you know protons are pumped across the membrane. Lecture 2310/31/12if housekeeping receptor mediated endocytosis, receptors may go to Golgi before going back to plasma membrane. Other vesicles may fuse with early endosome and vesicles from that compartment may exitand deliver receptors back to membrane. Those vesicles may go to golgi and either go to plasma membrane or cytoplasm. Other vesicles may bud from early endosome and deliver contents to intermediate compartments that eventually lead to lysosomal compartments. How do vesicles end up merging with one of those membrane compartments and not any other?What brings up fusion?Vesicles do no move by diffusion, but by cytoskeletal elements like microtubules, using the motor proteins attached to them. Upon reaching target compartment, and will attach using tethering protein, like hooks until they merge. At neurosynapse, vesicles with signal/neurotransmitters. Upon signaling, will merge with membrane of neuron at terminus and then secrete signal/contents into synapse. – triggered by change in membrane potential. Process: Vesicle originally brought into cell by receptor mediated endocytosis, thus with a clathrin coat that has been shed. Vesicle sitting in cytoplasm still with receptor and ligand attached, this vesicle is meant to merge with a target membrane – this is the endosome. In addition to receptor and ligand, the vesicle has SNARE. SNARE- filamentous proteins. Will form complex with proteins on membraneV-SNARE: vesicular T-SNARE: target membraneWhen membranes are close enough, the SNAREs will twist around each other to make complex, and will bring vesicle and target membrane close enough to merge. SNARE: has G-Protein; active when with GTPwill change conformation when GTP hydrolyzes.Rab-GTP: (Rab is G Protein)When membranes are brought close together (vesicle and target membrane) – Rab-GTP will bind to filamentous, tethering protein. Establishes link that keeps vesicle in vicinity of target membrane. Tethering protein: long protein that dangles but has affinity for the target protein.Once membranes are close enough, T-snare and V-snare interact/tangle/twist around, will form 4 subunit rope-like/complex where they will twist. Twisting is energy releasing - Spontaneous reaction to bring membranes together. Water molecules will purge out/squeezed out of membrane for the 2 to fuse. Doesn’t take energy to twist, takes energy to separate!Don’t need to remember specific v-snare and t-snares .Proteins involved in contortions are recycled in another
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