Protein Synthesis/Sorting/Transport Flashcards
Live Cells and Fluorescent Antibodies
- antibodies linked to fluorophores bind to specific proteins
- fluorescent dyes label intracellular structures
- Hoechst stain binds DNA in nucleus
Fluorescent Activated Cell Sorting (FACS)
- cells pass through laser
- cells given charge proportional to degree of fluorescence
- cells separated by an electric field
Cell Cycle Analysis
- cells with replicated (G2) DNA = x2 Hoechst stain
- more cells in G1 b/c longest phase
Disruption of Cell Plasma Membrane (5)
- Mechanical homogenization (cells squeezed)
- Sonication
- Pressure
- Non-ionic Detergents – Triton X-100
- Hypotonic Solution (swell + rupture)
Differential Centrifugation
- pellet nuclei
- pellet mitochondria, chloroplasts, lysosomes, peroxisomes
- pellet PM, ER, large polyribosomes
- pellet ribosomal subunits, small ribosomes
- remove supernatant btwn each step
- increase centrifugal force to isolate organelles by mass
Equilibrium Density Gradient Centrifugation
- separate based on density
- gradient of sucrose
- organelles migrate to sucrose layer of own density
Detergents
- are amphipathic
Non-ionic – disrupt lipid bilayer
Ionic – denature, disrupts bonds (SDS)
SDS-PAGE
- polyacrylamide gel and negatively charged detergent
- SDS denatures hydrophobic side chain
- gives protein negative charge
- protein move through gel based on size
Molecular Weight and Relative Mobility
low mass protein = high mobility
high mass protein = low mobility
Western Blotting Prodcedure
- electrophoresis and transfer
- primary antibody recognize protein
- secondary antibody (with enzyme) recognize primary
- chemiluminescence substrate + enzyme = glow
Western Blotting Use
- quantify intensity of darkness in each band
- proportional amount of protein in sample
Protein Targeting/Sorting
- direct proteins to right destination (organelles)
- direct proteins to secretory/non-secretory
Cytosolic Ribosomes
- remain in cytosol
- targeted to intracellular organelles
- specific signal sequence
Ribosomes attached to ER
- reside in ER
- sorted to PM, Golgi, Lysosomes
ER Structure
Rough ER – ribosomes on tubules
Protein Movement Inside/Outside of Cell
- Experiment 1
- secretory proteins first enter ER lumen
1/2 cells with protease
- lumen intact + protein preserved
1/2 cells with non-ionic detergent + protease
- protease enter lumen + proteins degraded
Protein Movement Inside/Outside of Cell
- Experiment 2
- translocation + translation simultaneous
mRNA –> translation
add microsomes = no proteins in microsome
mRNA + microsomes –> translation in microsome
mature protein in microsome (remove protein target signal)
Rough ER Components
- Amino terminal signal sequence
- Signal-recognition particle (SRP) – bind to new protein
- SRP receptor (with GTP) – bind to SRP
- Translocon (hydrolysis GTP) – polypeptide enter lumen
- Cleavage site (signal peptidase) – cleaves signal sequence
Glycosylation in ER
- add sugar to proteins
- transfer oligosaccharide onto dolichol
- transferred to an Asparagine residue by oligosaccharyl transferase
Protein Folding in ER
- Chaperone BiP
- Calnexin + Calreticulin – recognize sugar, prevent misfolding
- PDI – prevent inappropriate disulfide linkages
Prevents misfolding or aggregation of new proteins
Protein Import into Mitochondrial Matrix
- Hsp70 = chaperone protein
- prevents misfolding
- ATP hydrolysis
Mitochondrial Matrix Targeting Sequences
- guide to mitochondria
- recognized by TOM 20 –> guides to TOM 40
- TIM 44 opens channel == enters matrix
- Hsp70
- Matrix protease – cleaves targeting sequence
