Protein Synthesis/Sorting/Transport

Question 1

Live Cells and Fluorescent Antibodies

Answer 1

• antibodies linked to fluorophores bind to specific proteins
• fluorescent dyes label intracellular structures
• Hoechst stain binds DNA in nucleus

Question 2

Fluorescent Activated Cell Sorting (FACS)

Answer 2

• cells pass through laser
• cells given charge proportional to degree of fluorescence
• cells separated by an electric field

Question 3

Cell Cycle Analysis

Answer 3

• cells with replicated (G2) DNA = x2 Hoechst stain

- more cells in G1 b/c longest phase

Question 4

Disruption of Cell Plasma Membrane (5)

Answer 4

1. Mechanical homogenization (cells squeezed)
2. Sonication
3. Pressure
4. Non-ionic Detergents – Triton X-100
5. Hypotonic Solution (swell + rupture)

Question 5

Differential Centrifugation

Answer 5

• 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

Question 6

Equilibrium Density Gradient Centrifugation

Answer 6

• separate based on density
• gradient of sucrose
• organelles migrate to sucrose layer of own density

Question 7

Detergents

Answer 7

• are amphipathic

Non-ionic – disrupt lipid bilayer
Ionic – denature, disrupts bonds (SDS)

Question 8

SDS-PAGE

Answer 8

• polyacrylamide gel and negatively charged detergent
• SDS denatures hydrophobic side chain
• gives protein negative charge
• protein move through gel based on size

Question 9

Molecular Weight and Relative Mobility

Answer 9

low mass protein = high mobility

high mass protein = low mobility

Question 10

Western Blotting Prodcedure

Answer 10

• electrophoresis and transfer
• primary antibody recognize protein
• secondary antibody (with enzyme) recognize primary
• chemiluminescence substrate + enzyme = glow

Question 11

Western Blotting Use

Answer 11

• quantify intensity of darkness in each band

- proportional amount of protein in sample

Question 12

Protein Targeting/Sorting

Answer 12

• direct proteins to right destination (organelles)

- direct proteins to secretory/non-secretory

Question 13

Cytosolic Ribosomes

Answer 13

• remain in cytosol
• targeted to intracellular organelles
• specific signal sequence

Question 14

Ribosomes attached to ER

Answer 14

• reside in ER

- sorted to PM, Golgi, Lysosomes

Question 15

ER Structure

Answer 15

Rough ER – ribosomes on tubules

Question 16

Protein Movement Inside/Outside of Cell

- Experiment 1

Answer 16

• 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

Question 17

Protein Movement Inside/Outside of Cell

- Experiment 2

Answer 17

• translocation + translation simultaneous

mRNA –> translation
add microsomes = no proteins in microsome

mRNA + microsomes –> translation in microsome
mature protein in microsome (remove protein target signal)

Question 18

Rough ER Components

Answer 18

• 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

Question 19

Glycosylation in ER

Answer 19

• add sugar to proteins
• transfer oligosaccharide onto dolichol
• transferred to an Asparagine residue by oligosaccharyl transferase

Question 20

Protein Folding in ER

Answer 20

• Chaperone BiP
• Calnexin + Calreticulin – recognize sugar, prevent misfolding
• PDI – prevent inappropriate disulfide linkages

Prevents misfolding or aggregation of new proteins

Question 21

Protein Import into Mitochondrial Matrix

Answer 21

• Hsp70 = chaperone protein
• prevents misfolding
• ATP hydrolysis

Question 22

Mitochondrial Matrix Targeting Sequences

Answer 22

• guide to mitochondria
• recognized by TOM 20 –> guides to TOM 40
• TIM 44 opens channel == enters matrix
• Hsp70
• Matrix protease – cleaves targeting sequence