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Bis 104 Final Part 1

Bis 104 Final Part 1 - Notehal BIS 104 Review Guide for...

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Unformatted text preview: Notehal] BIS 104 12-06-10 Review Guide for Final PART 1 Announcements: Do not forget your Scantron and #2 pencil for the exam on Tuesday The Final starts at 8 AM. Do not miss it! Review Sessions: Sunday Dec. 5 at 5-6pm Monday Dec. 6 from 6-7pm Both Sessions will be held in Kleiber Hall NOTES: This Cumulative Review will consist not only of material covered in Lecture, which may have been seen in my previous documents, but also material from the required reading in Molecular Biology of the Cell the 5th Edition (Garland Science @2008]. Material From Chapter 1: Endosymbiotic Theory Chapter 8 Material: Techniques used to separate out and examine proteins: SDS-PAGE: (Fig. 8.18 and page 517 in book) - SDS detergent binds to hydrophobic regions of proteinsé breaks open the cell - Forces the proteins to unfold into extended polypeptide chains and releases them from associations with other proteins and lipids - [3-mercaptoethanol can be added to break S-S bonds and free up peptides - proteins of similar size move through the gel at similar speeds - larger proteins move through gel very slowly (Fig. 8.19) - allows for separation by size IS‘ISESZSSSSISQIB palmary antibody: mutiny antibodies: fll rabbit anflhody marlin-coupled amlhodles 11l.3 . Mel-d again“ dlnmd Igalnn rabbit 3:: antigen A Indbcdh: 55.1 '- r maker 35.5 Immobillud : f m... _|_ i _L Vi ZI.‘ 11.4 ' : II 3.5 mm 9.13 Mulcrular Eivlvgy um: cm (0 Garland Scam: 200K} Western Blotting a.k.a. Immunoblotting: (Fig. 8.20) - after SDS-PAGE, label all proteins present with an antibody that is coupled with a radio isotope, detectable enzyme, or fluorescent - transfer proteins to a sheet of nitrocellulose paper of nylon membrane - drive proteins out of the gel with a strong electrical field, which transfers the proteins to the membrane - then soak the membrane with labeled antibodyé will “tag" protein of interest if present - indirect immunocytochemistry (Fig. 9.18)9 antibody markers with fluorescent dyes Cell Fractionation followed by Centrifugation 9 Used to identify where proteins of interest are located within the cell (Fig. 8.10 pp.510-511) Items in the Items in the pellet su . ernatant 1000xg speed Everything else but the Nucleus nucleus Notehal] 12,000xg speed Microsomes, vesicles, Mitochondria, ER and Plasma chloroplasts, membranes lysosomes, peroxisomes 50,000xg speed Cytoplasm Microsomes, vesicles, ER and PM Density Gradient Centrifugation: (Fig. 8.11 pp.511-512) 1. Take membranes and put on top ofa Sucrose density gradient (5%-20%) 9 as the components move, the component hits the same density gradient as the sucrose it will stop and create a thick band 2. The remaining component will continue to move and form a band elsewhere. The first band will be the ER and the second hand will be PM Proteins can be separated out by Chromatography I Protein mixture is passed though a column containing a solid porous matrix I Can separate proteins by: . Charge (Ion-exchange)9 charged beads are packed into a column forcing proteins to fractionate according to charge . hydrophobicityé beads connected to hydrophobic side chains will slow down proteins and make them “stick" . Size (gel-filtration)9 column is filled with tiny porous heads 0 molecules small enough to enter the heads will linger inside 0 larger molecules will pass through quickly and emerge first affinity (Fig. 8.1409 more precise than other methods 0 takes advantage of biologically important interactions: antibody affinity, DNA affinity, sugar affinity Material From Chapter 9: Pulse-Chase Experiments: (Fig. 9.38 page 602) - used to study protein import - traces molecules with radioisotopes - used to prove whether or not a protein was inside the mitochondria - take a cell culture and add radio-labeled amino acid (AAs) (the pulse) i.e. 358-Met9 only exposed for 1-2 minutes - all the proteins synthesized in that time will use the radio-labeled AA - to stop process, culture is washed with cold methionine (Chase)9 exposed for 30-40 minutes - isolate mitochondria via cell fractionationé remove pellet and measure radio activity - Radio Activity measured in 3 ways: I No change in amount of radio activity compared to control% suggests nothing happened I Reduced amount of radio activityé reduction in protein amount. Los of protein Notehafl . Increase in radio active counté protein has entered the organelle of interest Confocal Laser-scanning Microscopy: (Fig. 9.20) - Similar to fluorescent microscopy - Pinpoint a specific point on the specimen instead of just passing light through the whole specimen at a specific depth - A pinhole aperture is placed at a position that us Confocal to the illuminated point. - Light from this point converges on the aperture and enters the detector - Allows you to look at specimens with finer in-phase detail and create a 3D image (Fig. 9.21) Calculating Resolution: D: 0.61 A/ n Sine (Fig. 9.6) The Light Microscope. (Fig. 9. 3) Limit of Resolution: 0. 2um - The more refracted light you can collect the better the image - If you put a sample in glass or water, refractive index: 1.3 - If you put a sample in oil immersion, refractive index: 1.4 - Depending on which objective you use, you can increase the resolution - Using a shorter wavelength of light like violet you can get better resolution9 close to the maximum - Can resolve about 1000x better than the naked eye by using a light microscope - Oil Immersion Lensé most animal cells viewed this way (Fig. Q9.1) Electron MicroscopylEMl (Fig. 9. 32)/ (Fig. 9. 4-2). specimen stained with electron dense material - Biological specimens must be “fixed" and stained due to the intensity of the vacuum - Uses a beam of electrons to pass through the specimen instead of light - Can make the wavelength of electrons very small by increasing the velocity of the voltage provided to the electrons - Increases the resolution to 0.002nm9 not able to ever achieve resolution. Can only achieve about an9 100x better resolution than the light microscope (Fig. 9.2) - CAN SEE: golgi, ER, details of the mitochondria and chloroplasts Scanning Electron Microscopy lSEM) (Fig. 9.4-9 pp.607-608): - Resolution up to 20nm (Fig. 9.48) - Used to look at the surface of an object9 cannot detect the interior of an object - Coat object with a thin layer of heavy metal - When scanned with a narrow beam of electrons, the surface with transmit electrons and expose a 3D structure of an object - Specimens can be: 0 Rapidly frozen 0 Fixed, dried, and coated with metal 0 Entire plants 0 Small animals9 can be put on the microscope with very little preparation Transmission Electron Microscopy (TEM) - can get higher resolution than SEM Notehafl - metal coating is sprayed obliquely so coating is thicker in some placesé metal shadowing - internal structures of cells can be imagedé freeze fracture I samples are rapidly frozen I cracked open with a knife I the ice level is lowered by sublimation of ice in the vacuumé freeze drying Phase Contrast Microsco e PCM Differential Interference Contrast Microsco e DICM :(Fig. 9.10) - Live cell imaging - PCM creates a contrast by using both the straight light and the refractive light (Fig 9.4) - Problems with PCM: the refractive index change is very high. Creates a halo around cells. - DICM creates a sharper imageé eliminates the halos (Fig. 9.11a and Fig. 9.11b) Dark field Microscopy: - illuminating rays of light are directed from the side - only scattered light enters the lens - allows viewing of cell processes like mitosis and cell migration Bright field Microscopy: - light passes through cell culture directlyé flooding it with light See Fig. 9.8 to compare images Fluorescence Microscopy: (Fig. 9.13) - more accurate than PCM - can use fluorescent dyes to label parts of a cell - Absorb shorter wavelength of light, but emit a longer wavelength of light (Fig. 9.14) - Differences from Light Microscope: I Very powerfully-sourced illuminating light is passed through two sets of filters . One filters the light before it reaches the specimen9 passes along only wavelengths of light that excite the dye ° The other filters light from the specimené passes along only wavelengths of light that are emitted by the dye C_p—ha ter 10: Membrane Structure: Properties of Phospholipids: - Consists of a polar head (Hydrophilic) and non-polar tails (Hydrophobic)9 amphiphillic - One chain of the lipid tail is saturated Notehal] - The other chain of the lipid tail is unsaturated and kinked due to a ciS-double bondé this is important for membrane fluidity - spontaneously form bilayers in water - forces water molecules to form an “ice-like" cage around them (Fig. 10.6) - they cluster together so that the least number of water molecules is affectedé energenically favored - to reduce interaction with water molecules the hydrophobic tails will aggregate to bury themselvesé form micelles or bilayers (Fig. 10.7) - hydrophilic heads remain exterior towards the water (Fig. 10.2) polar imfl‘fl’ m " '°‘ Animal Cells have 4 ma'or hos holi ids Fi . 10.16 : - $ - Phospholipids are n_ot evenly distributed ' 1 2 37° 5° between the 2 halves of the Plasma {w in: Membrane (PM) E: l: - Outer layer primarily consists of: 1. E E E Sphingomylein rilyT-zrllobm i 5: bay?” 2. Phosphotldyl chollne ml: 2 t: R \ - Inner layer primarlly con51sts of: 1. PE " g": 2. PS 5\ 5:: . - Asymmetrical distribution is critical in cell i :2: 1%. signaling, cell death (apoptosis) and converting extracellular signals to intra cellular signals - Some lipids are also glycolipids (Fig. 10.27) I Used in cell-recognition I Also contributions to the carbohydrate layer around the cell (Fig. 10.28) 9 protects the cell against mechanical and chemical change 9 keeps other cells at a distance to prevent unwanted protein-protein interactions Cholesterol and its role in membrane fluidity: - oriented between phospholipids such that the polar —OH group is next to the hydrophilic head (Fig. 10.5) - tightens the packing of lipids in the bilayer, but also increases fluidity by keeping hydrocarbon chains from tangling together - composition I cholesterol packaging I glycolipids - temperature I harder to freeze if the membrane is unsaturated I kinks make it more difficult for the hydrocarbon chains to pack togetheré membrane stays fluid (Fig. 10.12) h r11M ril:MmrnTrn r: - Passive Transport: high concentrationé low concentration 0 Channel Proteins: O Notehal] I Facilitated diffusion I Very fast diffusion I Creates a pore in the PM Transporter Proteins: I Requires a conformational change (Fig. 11.5) I Binds to a specific solute - Active Transport: requires energy to push solutes against gradients. Low concentrationé high concentration I Coupled Transporter (Fig. 11.7) 0 Couples the uphill transport of a solute with the downhill transport of another solute o Symporters (co-transporters) and Antiporters (exchangers) I ATP-Driven pumps: (Fig. 11.14) 0 Couple uphill transport with ATP hydrolysis 0 Examples of Pumps: Na+/K+ pump (Fig. 11.15), H+/K+ ATPase, and Ca++ ATPase (Fig. 11.13)9 found in skeletal muscle cells to cause muscle contractions I Light Driven Pumps (Fig. 10.33) 0 Contains chromophoreé gives protein a purple color 0 When activated, chromophore changes shape and causes a conformational change in the protein bacteriorhodopsin o Transfers one H+ out of the cell - Ion Channels and Gated Channels: 0 Can fluctuate between open and closed 0 Most commonly permeable to K+ 0 Ion selective o Mechanically Gated Channel: I Opened by movement, touch or pressure 0 Ligand-binding Channels: I Opened by binding ligands I Can be intracellular or extracellular (think of receptor proteins) 0 Voltage-Gated Channels: I Open up permeability to Na+ I Leads to the generation of action potentials in neurons I Automatically closes over time voltage- ligand-gain! ligand-gm mechanically gated (mellular (Intracellular gated :11: W R! W 1 '1' ‘1’ 0.. in M 9! "m ligand] ligand! (Fig. 11.21) Notehafl Chapter 12: Intracellular Compartments and Protein Sorting Three types of Sorting in the Cell (Fig. 12.6) 1. Gated Transport: operates similar to gated channels I Cytoplasmé nucleus I Transported through Nuclear Pore Complex (NPC) I Operate passively, but limited due to size of the pore 2. Transmembrane Transport: I Transported across a membrane i.e. ER, Mitochondria, Chloroplast I No pore in Mitochondria or chloroplast forcing proteins to go through the membrane I Mitochondria and Chloroplasts have two membranes 3. Vesicular transport: (Fig. 12.7) I ERé Golgi I Breaks Part of the membrane containing the protein off into a bud I Vesicle travels to recipient and fuses to its membrane, which releases the protein I Does Not pass through any membranes View Table 12.3 (P.702) For Sorting Signal Sequences Function of Signaling Sequences Example of Signal Sequence Import to nucleus PPKKKRKV **LYSINES ** Export from nucleus LALKLAGLNI **Leucines** Import to mitochindria N-terminus **Arginines** Import to plastid N-terminues **Serines** peroxisomes C-terminus **S-K-L** Im ort to ER LLLVGIL **Leucines** Sta in the ER C-terminus K-D-E-L, H-D-E-L Note: if protein has the N-terminal hydrophobic proteins (i.e. I and L) and internal KKRKKK sequence, protein will go to the ER due to co-translation (Fig. 12.35) Nucleus Transport: (Fig. 12.8 and 12.9) - Some proteins are shuttled constantly between nucleus and cytoplasm (Fig. 12.18) - Can have a sequence that allows them to go in the nucleus, but also out of the nucleus - Transcription Factorsé are phosphorylated to stay out of the nucleus, but then are bound by a phosphatase that activates the protein inside of the nucleus - All proteins going to the nucleus have to go through the Nuclear Pore Complex Nuclear Localization Sequence (NLS) (Fig. 12.11) I Responsible for the selectivity of the active nuclear import process I Determines if a protein can go inside the nucleus I Signal sequence usually consists of: KKKRK / lysines and arginine Nuclear Export Sequence (NES) I Determines if a protein will go out of the nucleus RAN GTPase Imposes Directionality of Transport through NPC - Ran is found in both the nucleus and cytosol - Required for import and (Fig. 12.14) - Has two conformation states9 dependent - GAP: (GTPase activating protein) triggers ATP hydrolysis and converts Ran-GTP9 Ran-GDP - GEF (Guanine exchange factor) converts Ran-GDPé Ran-GTP - The gradient of Ran's two conformations drives the direction of nuclear transport (Fig. 12.15) - Repeats of “PG" (Phenylalanine-glycine sequences) along fibrilsé proteins attach to FG's and bounced from one to the next until it hits the nuclear interface - When solutes reach the NPC, Ran-GTP binds to them releasing them from they transport molecules into the nucleus - The now empty import transporter with Ran-GTP bound is transported back out to the cytosol. - Once in the cytosol. Ran-GTP goes to Ran-GDP and is released from the transporter (Fig. 12.16) prof-in with nuclear cam delivered :4: (ml Innlizl‘linn signal 0» nuclear \5 export recepuor " «momma WWMMM mnuduus emnslmIaI Import of Proteins to Mitochondria: . . .. ._ . _ . . . . mammm .NUCLEIIIEXPOII‘I‘ - Protein Translocators in the mitochondrial membrane (Fig. 12.23) I Outer mitochondrial membrane translocatoré TOM complex I Inner mitochondrial membrane translocatoré TIM 23 complex I Each complex consists of a m - number of proteinsé contain "mm receptors that target "‘3,.'°.."..".“" signaling sequences __ _ _ i tween. - Import of Proteins (Fig. 12.25) . I Protein is synthesized then “mm": transported to organelles via 5m signal sequenceé unfolded . I Chaperones keep protein ' ' "mm SPACE from folding before it enters the mitochondriaé cytsolic I“ — _ Hsp70 (Fig. 6.86) I Sequence is recognized by receptor in TOM complex9 using ATP hydrolysis, removes the cytsolic Hsp70 (Fig. 12.26) Notehall I TOM complex threads protein through the TIM 23 complex I Mitochondrial Hsp70 hydrolizes ATP to pull protein through the TIM 23 complex I Folding initiated by mitochondrial Hsp 60 (Fig. 6.86) Protein Sorting into the ER: (Fig. 12.35) - Proteins programmed for outside of the ER are synthesized in the cytoplasm (Fig. 12.41a) - Proteins programmed for the ER are made on the ER membraneé done via co- translation ER rotein si nal is Guided: - Signal Recognition Particle (SRP) (Fig. 12.39a) - Signal sequence binds to SRP - SRP attaches to the ribosome and is received by the STP-receptor (Fig. 12.39b) - Protein is then through the translocator (Fig. 12.40)9 similar to mitochondria . § P} ER Translocator (Fig. 12. 42) “M ER has a pore called the Sec61 complex (Fig. 12. 42)9 four of these make up the translocator - Highly conserved from bacteria to eukaryotic cells - Pore can open and close - Proteins bind to the translocator, which opens it (Fig. 12.45) - Signal sequence is then clipped and the pore closes - Transmembrane Proteins: (Fig. 12.47) I has a start-transfer sequence and a stop- transfer sequence I binds to the translocator and is threaded through until the translocator hits the stop-transfer sequence I once the tranlocator reaches the stop-transfer m min? add numb." sequence, the protein is discharged laterally into the ER lipid bilayer hydrophobic hydmphilic ER Resident Proteins Modify proteins (Fig. 3.28) - Always have KDEL or HDEL sequences - Protein Disulfide isomerase (PDI) I Involved in 5-8 bonds and folding I S-S bonds can form between different polypeptides I Forms between cysteines I CXXC sequenceé determines S-S bond formations Multiple PDIs (human 8-10, plants ~16] - N-linked glycosylation (Fig. 12.50) 2 types of sugar added 1. N-linked 2. 0-linked9 only occurs in the golgi Site of glycosylation9 determine by sequence NXST 0 Not all asparagine is glycosylated Cytosolic proteins rarely have NXST sequence ° Glycosylation protein is only in the ER lumen Notehal] Precursor oligosaccharide gets attached to sequence (Fig. 12.51) As polypeptide is made, oligosaccharide attaches to NSXT Calreticulin/Calnexin cycle .. .mm-W MM” UDP unp- GEMPSGtWri GEM Guam ”arid-cm ERM” HIESBGWLE ”E; \«53 W“ gm“!!! 5.9mm? \ r M£rl|fi ens-ass . anus? g= ' fin-rm"- cm . Gum \ /,.EH. mam: : mmmm. " " CFTR Chapter 13 Material: Vesicular Transport: Organelle of Origin Vesicle Type Destination ER Cop II Golgi GOLGI Copl and Clathrin Coated Back to ER or to lysosomes PLASMA MEMBRANE Clathrin coated Endosome late endosome early O endosome f ‘\ P /' ‘5...“ / O-—+ \’ — y — a...” Cop 1 Vesicles: ‘04 \q‘ -.. trans Golgi plum cisternae network I—l Golgi apparatus secretory membrane vesicle Fig. 13.5 Notehal] - transport materials back to the ER from the golgi that were transfered out of the ER unintentionally9 ER resident proteins (Calnexin and calreticulin) - ER membrane proteins with C-terminal KKXX sequence bind to Cop1 components - C-terminal KDEL sequence9 recognized by receptor and shipped back to ER - Also used in golgi transport from ciS-golgi through the cisternae to the transgolgi - Assembly triggered by Arf Proteins CopII Vesicles: - Found in the ER - Carries ER products from ER9 Golgi - Assembly triggered by Sar1 Protein (Fig. 13.13a) Sarl- GDP' 1s inactive in the cytoplasm I Sarl- GEF converts Sar1- GDP (inactive)9 Sar1-GTP (active) I Sarl-GEF is present in ER membrane I Amphipathic helix of Sarl gets bound in the ER bilayer I Once Sarl is activated, Sec23 binds to it I Sec23 binds to Sec24- I Sec24 binds to cargo receptor I Cargo receptor binds to cargoé makes membrane curve I Sec13/Sec31 assist in CopII coated-vesicle formation9 buds off and forms cage similar to Clathrin - bud off from regions ofthe ER with no ribosomes - fuse together to form vesicular tubular clusters before reaching and fusing with the cis-golgi (Fig. 13.22 and 13.23) Clathrin-Coated Vesicles (Fig. 13.7) naked llama“! COAT ASSEMBLY BUB VEICLE ANDCMGO SELECI'KIN FORMATION SORIIMON UNCOA‘I'IHG - usually carries cargo...
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