Week 3 Lecture Notes
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This 11 page Class Notes was uploaded by Luke Holden on Sunday September 11, 2016. The Class Notes belongs to BIOL 4610 at Clemson University taught by Susan Chapman in Fall 2016. Since its upload, it has received 61 views.
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Date Created: 09/11/16
Week 3 Lecture Notes (9/6/14-9/8/14) The Nucleus One of the prominent spots in the cell Structure of Nucleus: o 2 Membranes around the nucleus Inner space between the two monolayers – Lumen/perinuclear space Outer membrane continuous with the ER and other cytoskeletal structures can be bound Tubular envelopes project into the nucleus Intermediate filaments called nuclear lamina (made from laminins) line the inner wall of the nucleus in a mesh like fashion Nucleoskeleton (nuclear matrix) that is an insoluble network that helps the nucleus maintain a dense structure Holds the chromatin in an organized fashion so that it can be expressed. Chromatin has its own location – chromosome territory o Nucleolus Functions in assembling the ribosomal subunits in eukaryotic cells No nucleolus means no life NO ACTIVE RIBESOMES IN THE NUCLEUS Signal recognition assembly (order tags) Protein Targeting to the Nucleus o No translation in the nucleus o Must be transported into the nucleus Pore Complexes takes care of this o Nuclear Localization Signal (NLS) Directs proteins into the Nucleus Very Specific 7 Positive (basic) residues Localized to the C-terminus Mechanically integrate with the rest of the cell Change one amino acid then you change the destination of the protein Example: Pyruvate Kinase Nuclear Pores o Pores are like corks that keep the cytosol of the nucleus from the cytosol of the cell o Line with the Nuclear Pore Complex 30 different proteins called nucleoporins symmetrical has central granular called the transport and moves solutes across the membrane In charge of the in and out flow Works by way of diffusion o Structure: Huge 12.5 millio daltons Spans both membranes Has selectivity filter Think of the structure of a simple pore like a rope hole in a tarp Nuclear Import via Importin/Ran dependent o Steps: Importin: Binds to the NLS on the protein and mediates it way into the nucleus Transported into the nucleus via the importin-protein complex The importin brings it into the nucleus and releases the protein and bind to Ran Then the Ran-GTP complex goes back out of the cell through the NPC Then importin dissociates by the hydrolysis of GTP Repeat Nuclear Import Ran independent o Mediated by Ca o Not much to know here Nuclear Export: o Similar to import o Used for RNA molecules o Can be Ran independent or Dependent o You have Nuclear Export Signals (NES) o Target proteins and the bound RNA for export o NES recognized by exportins o Dependent: Exportin binds to Cargo at the EPS The exportin-cargo complexes binds to Ran-GTP complex They are transported out of the cell via the NPC by the NTF2( Nuclear Transport Factor) GTP is hydrolyzed via GAP and the cargo dissociates as well as the rest of the complex. Exportin and Ran-GDP come back across the membrane Ran-GDPRan-GTP via the enzyme GEF o Independent: NO RAN mRNA use this pathway Steps: o mRNa is bound by NXF1 and NX1 (these are chaperon proteins that prevent the mRNA from wadding up o Transportation through the nucleus o Rna helicase (Dbp5) comes and removes the chaperon proteins by hydrolyzing ATP o NXF1/NXT1 come back across the membrane using the Ran-Dependent import process o Ran-GTP gradient needed: GEF (guanine-nucleotide exchange factor) keep the Ran-GTP levels high inside the nucleus The cytosol contains GAP (GTPase activating Protein that promotes hydrolysis) The Endomembrane System Components: ER o Structure: Continuous network of flattened sacs tubules and vesicles through the cytoplasm of a eukaryotic cell Cisternane-membrane bound sacs ER lumen- space inside the sacs Rough ER Smooth ER- ribosomes on the cytosolic side of the lacks ribosomes and has others membrane form tubular structures Transitional elements make vesicles Functions in producing steroid that go back and forth from ER to golgi (resemble smooth ER) hormones tend to have extensive networks of smooth ER Form Large Flatened Sacs Drug Detox Cells with lots of secrartory proteins have prominent rough ER networks Carb metablosims Enzymes are involved with protein Ca storage synthesis. Steriod Biosynethesis Sited for: Processing and storing nonprotein initial steps of carbs to molecules within cells glycoproteins Folding of polypeptides Recognition and removal of misfolded proteins Assembly of multimeric proteins Quality Control Sends bad proteins out to be degraded by ERAD to proteasomes Secretory Pathway (ER-Ribesomes) Step 1: Import into ER o mRNA is sent to a free ribosome to be translated o Once the ER signal sequences comes out, it is bound by the SRP or (signal recognition particle) to the hydrophobic region SRP Made up of 2 Subunits FFA or p54 FtsY or RP recα GTP bound in protein Hydrophobic binding groove- binds to hydrophobic bing region o The binding of the SRP causes translation to stop and thus it moves towards the ER This is called DOCKING THE TRANSLOCON o The SRP binds the stopped protein complex to a SRP receptor in with it is attached to a translocon by hydrolyzing 1 GTP and the SRP dissociates and translation can continue through the translocon Translocon has a SRP and Ribosome Receptor It also has a pore protein that forms a channel to allow the polypeptide to enter the ER and signal peptidase removes the ER signal sequence o The channel opens o GTP is hydrolyzed again and the SRP releases o The protein folds in the Lumen of the ER A word about puromycin: o Completely stops all translation with the ER o This completely wipes out micro flora in the gut o Makes you extremely susceptible to C-diff This produces a toxin that binds to your villi Types of ER proteins Type 1- Put in membrane o ER recognition sequence o SRP-brings it to the ER o Stop transfer sequence is translated and the rest of the protein can’t go through o peptidyl transferase cuts off signal sequence. o N-terminus goes in first translocon will put it in the membrane Type 2- (backwards) Flip type 1 o N- terminus is on the cytosolic side Never had a signal sequence on that side Signal anchor seq is what got it down to the translocon Therefore, C-term goes in first Type 3- o No signal o The signal anchor is close to N-terminus o Similar to Type 1! Be Careful Tail Anchored o The signal is on C-terminus goes into Get3 o protein complex recog and brings to ER (binds get 1+2) o Moves C-End into lipid bilayer by hydrolysis of ATP Type 4- N terminus in exoplasmic space (Multi-pass) o Various stop transfer sequences cause the multiple passes Even #- C-terminus on the same side as the N-terminus Odd #- N-terminus and C-terminus on opposite ends GPI-Anchored Proteins o The GPI molecule has both a polar (N-terminus end which contains carbohydrate and phosphate residues) and nonpolar ends (C- terminus which contains fatty acyl tails) o GPI transmidase cleaves the nonpolar end which is in the membrane and only allows into to interact on one leaflet rather than both. And transfers the carboxyl group of the polar half of an amino group on the bilayer. o Bam GPI-anchored
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