Lecture Set 10

Question 1

How can you isolate organelles?

Answer 1

1) subcellular fractionation –> a) homogenize to disrupt plasma membrane, release soluble proteins, maintain organelle integrity (through blender, sonication, aspiration) b) differential centrifugation –> have different rates of sedimentation based on size/density
2) rate-zonal (density gradient) centrifugation –> type of differential centrifugation
sample layered on top of solution (sucrose gradient) eventually all things will sediment
3) equilibrium density –> organelles move in density gradient until they reach equilibrium

Question 2

What are transitional elements? what occurs to ER during homogenization?

Answer 2

subdomain of rough ER, helps form transition vesicles

during homogenization, ER forms microsomes (small vesicles)

Question 3

What are the 4 roles of smooth ER?

Answer 3

1) drug detoxification –> cytochrome P-450 –> hydroxalates compounds through oxygenases, or monooxygenases that incorporate oxygen atoms into product to increase solubility, leads to degradation
2) carbohydrate metabolism –> glucose-6-phosphatase removes phosphate from glucose to allow glycogen to cross membrane into bloodstream
3) Ca2+ storage –> SR
4) steroid biosynthesis –> acetyl CoA

Question 4

How does the ER form membrane lipids?

Answer 4

ER attaches fatty acid to glycerol phosphate, then phosphatase removes phosphate to from DAG, then head group is added

Question 5

What are the 2 types of translocation?

Answer 5

co-translational, post translational

Question 6

What are the 4 protein modifications in the ER

Answer 6

1) glycosylation –> only N-linked, attach oligosaccharide precursor. composed of 5 mannose, 2 GluNAc. oligosaccharide then flipped to other side through, transferred through dolichol phosphate
2) disulfide bond formation –> PDI (phosphate disulfide isomerase) fixes misformed disulfide bonds
3) folding, aggregation of proteins –> uses chaperone molecules or chaperonins
4) proteolytic cleavage

Question 7

what happens to misfolded proteins?

Answer 7

sent to cytoplasm for degradation through ERAD (ER associated degradation) protein
leads to UPR (unfolded protein response) and creation of more chaperones

Question 8

What are the two models of Golgi transportation?

Answer 8

1) vesicular transportation –> vesicles bud off back and forth
2) cisternal maturation –> vesicles from ER form cis-Golgi cisterna, then matures into medial, then becomes trans-Golgi, trans golgi forms vesicles for anterograde/retrograde transport

Question 9

What are 3 examples of modification occuring in the golgi?

Answer 9

1) O-linked glycosylation
2) glucan synthetases –> form oligosaccharides from monosaccharides
3) glyosyl transferases –> attach carbohydrate groups to proteins