Endocytosis and Protein Degradation

Question 1

Organelles get damaged and

Answer 1

need to be removed and replaced

Question 2

Proteins have a

Answer 2

finite lifetimes.

Question 3

Misfolded proteins will

Answer 3

create havoc if they are not destroyed.

Question 4

Both good and bad endocytosed materials

Answer 4

need to be degraded.

Question 5

Major Functions of the ER

Answer 5

1. Synthesis of lipids (phospholipid, ceramide, and cholesterol)
2. Control of cholesterol homeostasis (cholesterol sensor and synthesis)
3. Ca+2 storage (rapid uptake and release)
4. Synthesis of proteins on membrane bound
   ribosomes
5. Co-translational folding of proteins and
   early post-translational modifications
6. Quality Control

Question 6

Molecular chaperone proteins

Answer 6

1. Help fold a protein by binding to exposed hydrophobic patches in incompletely folded proteins (hsp70)
2. Form large barrel-shaped structures to act as an “isolation chamber” into which misfolded proteins are fed to prevent their aggregation and help it to refold (hsp60)

Question 7

ER Quality Control

Answer 7

1. Provide optimized oxidizing environment
   – Folding
   – Oligomeric assembly
2. Folding enzymes
   – ERp57- thiol oxidoreductase (allows formation of disulfide bonds)
   3 . Molecular Chaperones- ATPases
   – BiP- Hsp70 family
3. Folding Sensors/ Quality Control
   – UDP-glucose: glycoprotein glucosyltransferase (UGGT)

Question 8

Proteasome

Answer 8

(ATP-dependent protease that constitutes 1% of cellular protein)

Dispersed throughout the cytosol and nucleus

Question 9

Proteosomes also monitor the

Answer 9

monitors the ER (proteins detected to be misfolded are retrotranslocated back into the cytosol for degradation)

Question 10

Proteasomal degradation (ubiquitin mediated)

Answer 10

ubiqutiation-covalent attachment of ubiquitin (76 aa) to acceptor lysine E-amino group in protein to be degraded

Question 11

Proteolysis occurs within the

Answer 11

central chamber

Question 12

Only β-subunits are _____

Answer 12

proteolytically active”

Question 13

α-subunits regulate

Answer 13

substrate entry into the “death chamber””

Question 14

Substrate is “spiraled” through the chamber and cleaved by

Answer 14

different activities associated with different b-subunits; ~7-9 amino acid peptides are released;

Question 15

cleavage does not require

Answer 15

ATP;

Question 16

ATP required for

Answer 16

unfolding and translocation

Question 17

Degradation:

Answer 17

mechanisms that provide spatial control

Question 18

Proteasome-

Answer 18

degrades only proteins

– Polyubiquibitinated proteins (tetra-ubiquitin minimal targeting signal)

Question 19

Lysosome-

Answer 19

degrades all cellular components
– Targeted via the endocytic pathway
– Monoubiquitinated transmembrane proteins
– Regulated at the Multivesicular Body

Question 20

• Autophagy

Answer 20

– Direct transport into the lysosomal lumen from cytoplasm

Question 21

Lysosomes Degrade all

Answer 21

Macromolecules in an Acidic Environment

Question 22

Transporters in lysosomal membrane allow exit of

Answer 22

amino acids, monosaccharides, nucleotides, and lipids for reutilization within the cell.

Question 23

Lysosomal storage diseases are

Answer 23

• Metabolic disorders that result from defects in lysosomal function
• Specific enzymes are defective or deficient

Question 24

examples of lysosomal storage diseases

Answer 24

– Tay-Sachs:
– Gaucher’s disease:
– Niemann-Pick:

Question 25

– Tay-Sachs:

Answer 25

beta-hexosaminidase, breaks down gangliosides in neurons

Question 26

Gaucher’s disease:

Answer 26

beta-glucosidase, breaks down glucosylceramide in monocytes and leukocytes

Question 27

Niemann-Pick:

Answer 27

(1) sphingomyelinase, breaks down sphingomyelin in macrophages and
(2) cholesterol transporter, moves cholesterol from the lysosome to the cytosol.

Question 28

Two major routes fro small volume endocytosis

Answer 28

1. clathrin coated vesicles

2. calveolae

Question 29

Clathrin Coated Vesicles function to

Answer 29

Selectively sort cargo at the cell membrane, trans-Golgi network and endosomal compartments for multiple membrane traffic pathways.

Question 30

In clathrin coated vesicles, after a vesicle buds into the cytoplasm, the

Answer 30

the coat rapidly disassembles allowing the clathrin to recycle while the vesicle gets transported to a variety of locations.

Question 31

In clathrin coated vesicles, Adaptor molecules are responsible for

Answer 31

self assembly and recruitment.

Question 32

Calveolae arises from

Answer 32

invaginations on the surface of the cell o Look like “little caves,” hence their name

Question 33

3 major coat proteins for calveolae:

Answer 33

1. Calveolin 1 – located everywhere
2. Calveolin 2 – seem to work with calveolin 1
3. Calveolin 3 – specific to skeletal and cardiac muscle

Question 34

Calveolin 1 – located

Answer 34

everywhere

Question 35

Calveolin 2 –

Answer 35

seem to work with calveolin 1

Question 36

Calveolin 3 –

Answer 36

specific to skeletal and cardiac muscle

Question 37

Mutations in Calveolin 3 cause

Answer 37

muscle diseases such as Limb Girdle Disease and Rippling Muscle Disease

Question 38

Cholera toxin gets into the cell via _____ exclusively.

Answer 38

calveolae

Question 39

Dynamin pinches off both

Answer 39

clathrin coated vesicles and calveolae.

Question 40

The ER provides an optimized oxidizing environment for

Answer 40

folding and oligomeric assembly.

Question 41

The ER has folding enzymes associated with it such as ______

Answer 41

ERp57

Question 42

Erp57: is ____ and allows formation of

Answer 42

thiol oxidoreductase

disulfide bonds with the new protein to hold it in place with calnexin

Question 43

Calnexin has a _____ pocket

o ERp57 holds the protein in place so that it presents its

Answer 43

glucose

glucose molecule to the calnexin pocket:

Question 44

If the protein has been folded correctly, ______

If it has been folded incorrectly, it will_____

Answer 44

the glucose will fit in the pocket.

not fit in the calnexin pocket

Question 45

If incorrect, it will be sent to the

Answer 45

lysosome for destruction or else given a chance

to refold correctly.

Question 46

The ER has ATPase molecular chaperones-

Answer 46

ATPases such as the BiP- Hsp70 family

Question 47

Finally, the ER has Folding Sensors:

Answer 47

1. UDP-glucose

2. Glycoprotein glucosyltransferase (UGGT)

Question 48

two types of molecular chaperones.

Answer 48

1. Hsp70

2. Hsp60

Question 49

Hsp70:

Answer 49

Binds to exposed hydrophobic patches of incompletely folded proteins to prevent aggregation.

Question 50

Hsp60

Answer 50

Forms a large, barrel shaped structure that acts as an isolation chamber. Misfolded proteins are
fed into it to prevent aggregation and to promote proper refolding.

Question 51

Proteasome Structure:

Answer 51

1. 2 mega-Daltons in weight – very large
2. 26S proteasome made up of 3 macro-subunits o 19S cap on each end – regulatory region
3. Base of cap contains 6 “AAA-ATPases”
4. Lid unit of cap contains poly-ubiquitin recognition molecules o Central 20S cylinder – the actual active site of the proteasome
5. The proteasome is ATP-powered

Question 52

Beta subunits are ______

Answer 52

proteolytically active

Question 53

Ubiquitination:

Occurs in three steps:

Answer 53

1. E1 ubiquitin – activating enzyme
2. E2- ubiquitin – conjugating enzyme which takes activated ubiquitin and transfers it to E3.
3. E3- ubiquitin – protein ligating enzyme.

Question 54

β1

Answer 54

– caspase-like: cleaves after acidic amino acids

Question 55

β2 –

Answer 55

trypsin-like: cleaves after basic amino acids

Question 56

β3 –

Answer 56

cleaves after hydrophobic amino acids

Question 57

E3s confer______to ubiquitination.

Answer 57

specificity and regulation

Question 58

E1 ubiquitin –

Answer 58

activating enzyme

Question 59

E2- ubiquitin –

Answer 59

conjugating enzyme which takes activated ubiquitin and transfers it to E3.

Question 60

E3- ubiquitin –

Answer 60

protein ligating enzyme.

Question 61

E3 binds directly to

Answer 61

substrate and covalently attaches the ubiquitin to the substrate.

Question 62

Once a single ubiquitin is covalently attached to a protein, additional ubiquitins are linked preferentially to

Answer 62

Lys48 of ubiquitin to form a poly-ubiquitin chain.

Question 63

Ubiquitin is

Answer 63

highly conserved

Question 64

The lysosome degrades all cellular components

Answer 64

1. Digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria
2. Targeted via the endocytic pathway
3. Mono-ubiquitinated transmembrane proteins
4. Regulated at the Multivesicular Body

Question 65

The environment of the lysosome is.

Answer 65

acidic (pH 4.5).

Question 66

The lysosome maintains this pH differential by

Answer 66

pumping protons (H+ ions) from the cytosol across the membrane via proton pumps and chloride ion channels

Question 67

The lysosomal membrane protects the cytosol, and therefore the rest of the cell, from the

Answer 67

degradative enzymes within the lysosome.

Question 68

The cell is additionally protected from any lysosomal acid hydrolases that leak into the cytosol because

Answer 68

these enzymes are pH-sensitive and do not function as well in the alkaline environment of the cytosol.

Question 69

Transporters in lysosomal membrane allow exit of

Answer 69

amino acids, monosaccharides, nucleotides, and lipids for reutilization within the cell.

Question 70

two major routes of endocytosis:

Answer 70

1) phagocytosis and

2) pinocytosis or small vesicle formation.

Question 71

Caveolae

Answer 71

(little cavities) are small endocytic vesicles that form

without coat proteins.