Endocytosis and Protein Degradation

Question 1

1. Describe two major routes for small volume endocytosis.
2. Explain how quality control of protein synthesis is ensured in the ER.
3. Describe two types of molecular chaperones.
4. Describe the proteasome, protein degradation, and the role of ubiquitin.
5. Describe the functions of the lysosome.

Answer 1

x

Question 2

There are two major routes of endocytosis:

Answer 2

(1) phagocytosis

(2) pinocytosis or small vesicle formation

Question 3

What cell types carry out phagocytosis?

Answer 3

Phagocytosis in multicellular organisms is normally carried out by specialized cells in the blood–macrophages and neutrophils.

These cells recognize foreign organisms like bacteria, engulf them, and deliver them to lysosomes for degradation. Macrophages and neutrophils also recognize apoptotic cells

Question 4

___ of vesicles involves small volumes, and usually is associated with specific uptake of ligands and receptors.

Answer 4

Pinocytosis

Question 5

What are the three main protein degradation pathways?

Answer 5

1) ubiquitin-proteasome system (UPS)
2) the lysosome (mentioned above as part of the endocytosis pathway)
3) autophagy

T[he UPS is responsible for the rapid degradation of proteins when fast adaptation is needed, and UPS protein makes up about 1% of the protein in a cell. Autophagy is mainly involved in the degradation of long-lived proteins and entire organelles; it plays an important role during development and is required for the adaptation to environmental stresses such as starvation.]

Question 6

Besides not working as designed, what problem is caused by misfolded proteins?

Answer 6

They usually have hydrophobic domains now exposed to cytosol. These exposed hydrophobic domains often bind to hydrophobic domains of other misfolded proteins and form large aggregates inside the cell.

Question 7

What are the two examples of chaperone proteins given in class? In what way does each work? Bonus: why are they so named?

Answer 7

Hsp70 prevents aggregation by binding to exposed hydrophobic patches in incompletely folded proteins. appears to be co-translational

Hsp60 acts as an isolation chamber. Misfolded proteins are fed into the chamber to prevent aggregation and to help it to refold. GroES cap covers chamber.

[The two best examples of these are hsp70 and hsp60. Hsp stands for heat shock protein – these proteins are made in high abundance when cells are heat shocked]

Question 8

What mechanisms in the ER exist to ensure proper folding (3)?

Answer 8

1) ERp57 folding enzyme that allows formation of disulfide bonds
2) BiP chaperone which is an Hsp70-like protein that uses ATP to help proteins fold
3) folding sensors that monitor unfolded proteins and hold them in the ER until they fold properly or are shuttled to a degradation pathway (UGGT - see next)

Question 9

What is an example of a folding sensory pathway in the ER that prevents misfolding?

Answer 9

UGGT (UDP-glucose:glycoprotein glucosyltransferase)

Basically the protein is glycosylated until after it folds. When the sugar is attached, so are calnexin (CNX) and calreticulin. When the sugar is cleaved, C and C disengage and if the protein is properly folded it goes on it’s merry way. If not, it is re-glycosylated and the process repeats. After x number of failed attempts, it is retrotransported, ubiquinated, and destroyed.

Question 10

What is ubiquitin?

Answer 10

Ubiquitin is a 76 amino acid protein. A chain of at least four ubiquitins is required by the proteasome as a tag for degradation.

[It is remarkably conserved in all cells. The sequence is the same in flies and humans, and 74 of 76 amino acids are identical between plants and animals. Multiubiquination (<poly) can be used as a regulatory mechanism ie histones–not all ubiquitination initiates degradation]

Question 11

Ubiquitination requires 3 proteins. What are they called and what does each do?

Answer 11

E1: activation
E2: conjugation
E3: ligation and chain extension (puts the poly)

Question 12

Describe the proteosome.

Answer 12

The proteosome is a Beta sandwiched on alpha bread. The Beta subunits cleave, the alpha subunits lead proteins to the butcher. Ha, B for Butcher. See what I did just then?

aaa
bbb
bbb
aaa

[Substrate is “spiraled” through the chamber and cleaved by different activities associated with different b-subunits; ~7-9 amino acid peptides are released; cleavage does not require ATP; ATP required for unfolding and translocation]

Question 13

Describe the cleavage. (Bonus card)
b1:
b2:
b5:

Answer 13

b1 - caspase-like: cleaves after acidic aa
b2 - trypsin-like: cleaves after basic
b5 - chymotrypsin-like: cleaves after hydrophobic

Question 14

The proteosome degrades ____.

Answer 14

ONLY proteins polyubiquibitinated proteins

Question 15

Lysosome degrades _____.

Answer 15

All cellular components.

Question 16

Lysosomes detail.

Answer 16

degrade extracellular materials taken up by endocytosis and some intracellular components. Lysosomes contain many enzymes that together will degrade all classes of molecules – proteins, lipids and sugars. The lumen is acidic (pH 5) due to the activity of a proton pump.