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Techniques and protein synthesis, transport and sorting, step 1, step 2,…

- - - - fluorescent activated cell sorting (FACS)
        
        process
        
        cells pass a tube to a laser in a line
        
        laser excites the fluorophores
        
        emitted light goes through filter and hits fluorescent light detector
        
        filter works to only use the optimal excitation wavelength
        
        cells pass through charged plates giving them a charge based on the amount they fluoresced
        
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        at this stage, the intensity of red and green fluorescence can be recorded and can calculate proportion of each cell population
        
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  - - - high intensity of staining means cell is in the G2 stage
      - more cell are in G1 compared to the G2 stage because cells do not divide at same time and G1 is the longest phase
- - - - force cells through a narrow valves to break the membrane
    - - selectively deteriorate the membrane
    - - use ultrasound in quick pulses to ONLY disrupt the membrane (more aggressive uses destroys ALL membranes)
    - - causes cells to swell and burst due to low salt concentration solution
    - - crush the cells
        
        use centrifugation to process the cell
        
        use differential centrifugation to separate the homogenate (intracellular parts of the cell)
        
        process repeats by separating supernatant and centrifuging it (liquid portion) until only cytosol left (must do process from larger to smaller organnelles)
        
        larger organelles can be separated by smaller g and smaller organelles need a higher g
        
        separating the pellet (equilibrium density-gradient centrifugation)
        
        useful if want to extract an organelle out of the pellet
        
        the separation is done based on density
        
        organelle is resuspended in solution and placed on a tube with increasing sucrose density
        
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        the rotor must be balanced and spin the contraption in a vacuum to avoid friction with air (causing sample to warm up)
- - - - disrupts only the bilayer
        
        can then separate proteins with SDS page
        
        proteins first denatured and all get a net - charge by using SDS
        
        proteins move from cathode (- charge) to anode (+ charge)
        
        high MW proteins move slower while low MW proteins move faster
        
        how to detect the protein
        
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        on the SDS page there is an M lane with know MW that is used to compare and determine the Mw of the other proteins
        
        there is a buffer above and below the gel
    - - disrupts the bilayer AND H-bonds of the proteins due to ionic portions
      - they are amphipathic such that the hydrophobic domains can penetrate through the membrane
- - - - sorting
        
        directs proteins to 2 pathways
        
        secretory pathway
        
        protein is sent to the membrane or is secreted
        
        non-secretory pathway
        
        protein eventually goes to lysosomes
        
        requires ER signal sequence tp go to ER membrane
        
        requires targeting sequence to go to other organelles
      - this stage directs the protein to the right location in
        cells
        
        this occurs during or after the synthesis of the protein (aka translation)
  - - - ribosomes in the cytosol
        
        proteins can be non-targeted
        
        remains in cytosol
        
        proteins can be targeted
        
        they have a specific signal sequence
        
        guides them to cellular organnelles
      - ribosomes on the rough ER
        
        remains in the ER
        
        directed to the PM, golgi or lysosomes
  - - - Has cisternae in folds and vesicles separated from the cytosol
      - has ribosomes on it
      - it extends from the nucleus
    - - prove secreted proteins must enter the ER for processing
        
        lyse cell
        
        homogenize ER to create microsomes and replicate a functional ER
        
        treat with non-ionic detergent and proteases
        
        no proteins were detected because all chewed up
        
        treat with proteases only
        
        proteins are detected
      - prove translocation and translation occur at the same time
        
        take translational apparatus and peptide chain with signal sequence on N-terminus
        
        add microsomal membranes
        
        no proteins were incorporated inside
        
        take translational apparatus and microsome
        
        found that proteins were found inside and their signal sequence was chopped off
  - - - to bring them them mitchondria, uses Hsp 7 and ATP to prevent misfolds
    - - has a matrix targeting sequence and Hsp70
        
        the seq is recognized by the import receptor, tom 20
        
        tom 20 moves the peptide to tom 40
        
        tom 40 opens and allows peptide to go through
        
        tim 44 works with tom 40 to get the peptide inside the matrix
        
        they work together at wavy top parts of the mitochondria
        
        in the matrix, the matrix Hsp 70helps the protein fold
        
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- - - - formed from the initial translation
    - - guide the translational apparatus complex to the SRP receptor binding to the signal seq
    - - where the SRP goes
    - - protein channel that allows the peptides to feed through the ER
    - - where the signal peptide is cleaved
  - - - SRP binds to the SRP receptor using GTP (energy)
        
        the translocon opens and the peptide goes through as translation resume (pushes the sequence in)
        
        at same time SRP dissociates from SRP receptor through the hydrolysis of GTP
        
        the signal sequence is cleaved by the signal peptidase
        
        stop codon is reached and protein is folded in the ER
        
        the ribosome moves to the translocon
  - - - addition of a sugar to the peptide chain
        
        these proteins are usually in proteins that function at membrane or are secreted (secretory)
        
        proceeds with transferring a 14-residue oligosaccharide precursor from dolichol to the peptide
        
        it is usually added on the asparagine side of the nascent peptide using the enzyme oligosaccharyl transferase
        
        glycosidases work to modify by chopping some of the sugars off
      - helps with protein folding and protein stability
    - - formed with chaperon protein PDI
        
        prevents the misfold by covalently binding to the cysteine residues
        
        guide the protein to form proper cysteine linkages
    - - hydrophobic proteins are prone to misfold in the ER
        
        chaperones to help prevent misfolds
        
        Bip
        
        prevent the self-association of the protein
        
        lectins(calnexin and calreticulin
        
        prevent misfolding by binding to sugar residues
        
        when the protein is folded properly, it is transported from the ER to the golgi