Lecture 8 - endomembrane system part 3

Question 1

do membranes form de novo?

Answer 1

no, all membranes form from pre-existing membranes

Question 2

Where are most membrane proteins and lipids synthesized?

Answer 2

ER, with the exceptions of glycolipids synthesized in the golgi

Question 3

How are nascent ER membrane proteins and lipids distributed and or orientated in lipid bilayer?

Answer 3

asymmetric manner

Question 4

Where are integral membrane proteins?

Answer 4

different regions of protein located on either cytoplasmic or exoplasmic

Question 5

Where are peripheral membrane proteins located?

Answer 5

located on either cytoplasmic or lumenal side of ER membrane

Question 6

Where are membrane phospholipids located?

Answer 6

distributed unequally between cytoplasmic and exoplasmic leaflets of ER membrane bilayer

Question 7

What type of asymmetry is established at the ER?

Answer 7

asymmetry and maintained throughout rest of endomembrane system

Question 8

Are cytoplasmic and exoplasmic faces of cellular membranes conserved throughout the endomembrane system?

Answer 8

yes

Question 9

What are the four final steps in co translational translocation pathway?

Answer 9

1. signal sequence cleavage - removal of n-term signal sequence by signal peptidase
2. initial stages of glycosylation - covalent addition of unique carbohydrate s.c. to specific AA of nascent protein (required for proper folding. protein-protein binding)
3. protein folding and assembly - nascent protein folded into proper 3D conformation and oligomeric assembly by molecular chaperones (reticuloplasms)
4. protein quality control - misfolded and/or improperly assembled proteins recognized and degraded

Question 10

What does the ER serve as?

Answer 10

a quality control site for nascent proteins since represents first compartment in endomembrane system

Question 11

What are most proteins synthesized in the ER?

Answer 11

glycoproteins - linked to one or more sugar chains

Question 12

What do sugar groups do for the protein?

Answer 12

aid in proper folding and serve as a binding site for other macromolecules that interact with the protein?

Question 13

What is the most common type of glycosylation?

Answer 13

N-linked glycosylation

Question 14

What is N-linked glycosylation?

Answer 14

addition of specific short chains of sugar monomers (linked together in specific order to form oligosaccharide) to terminal amino group of asparagine (N)

Question 15

What are the two stages of N-linked glycosylation?

Answer 15

core glycosylation
core modification

Question 15

What is core glycosylation?

Answer 15

various ER membrane-bound glycosyltransferases synthesize core oligosaccharide

Question 15

What does tunicamycin do?

Answer 15

blocks first step of N-linked glycosylation preventing proper folding of nascent ER proteins

Question 15

What are the steps to core glycosylation?

Answer 15

1. addition of first sugar to dolichol phosphate
2. glycosyltransferases continue to add sugars at specific positions on growing core oligosaccharide
3. transfer of core oligosaccharide from dolichol lipid carrier to nascent soluble membrane protein while being synthesized (empty dolichol phosphate recycled)
4. core oligosaccharides transferred to lumenal acing portions of nascent ER proteins with specific AA sequence motif: -N-x-S/T-

Question 15

What is dolichol phosphate?

Answer 15

membrane lipid serving as membrane anchor and carrier for new growing core oligosaccharide

Question 16

What occurs during core modification?

Answer 16

• attached 14-sugar core oligosaccharides sequentially trimmed and modified
  2. two of 3 terminal glucose units removed (trimmed) by ER lumenal glucosidases
  3. subsequent removal and re-addition of last glucose unit important for proper protein folding assembly
  reticuloplasms and PDI bind to nascent glycoprotein
  ER lumen glucosidase removes last glucose unit from core oligosaccharide during latter step
  nascent protein released from reticuloplasmins
  4. one mannose unit removed by ER lumen mannosidase

Question 17

What proteins mediate N-linked glycosylation?

Answer 17

reticuloplasmins
protein disulfide isomerase (PDI)

Question 18

What are reticuloplasmins?

Answer 18

ER molecular chaperones, including BiP, calreticulin and calnexin
bind transiently to nascent ER proteins while being synthesized via sec 61 cotranslational translocation pathway to prevent misfolding or aggregation

Question 19

What is protein disulfide isomerase?

Answer 19

catalyzes formation of intra/intermolecular disulfide bonds

Question 20

What do disulfide bonds between cysteine residues on nascent proteins do?

Answer 20

promote proper folding and assembly by stabilizing their 3D conformation

Question 21

What does adding the core oligosaccharide to the nascent protein do in N-linked glycosylation?

Answer 21

also contribute to proper protein folding assembly and stability and participate in protein quality control

Question 22

What does the nascent glycoprotein do once assembled through N-linked glycosylation?

Answer 22

functions as ER resident protein or transported via vesicles from ER to golgi where n-linked glycosylation continues then resides in golgi or moves to other compartments in endomembrane system,

Question 23

What happens if the nascent glycoprotein is misfolded/misassembled?

Answer 23

recognized by UGGT monitoring enzyme, it addes back a single glucose unit to the oligosaccharide core and then the misfolded protein binds again to calnexin, calreticulin and BiP to mediate proper protein folding again the process is repeated until the protein is properly folded and assembled

Question 24

What is UGGT monitoring enzyme?

Answer 24

glucosyltransferase - serves as protein conformation sensing protein. recognizes hydrophobic residues usually masked inside correctly folded protein

Question 25

What is the ERAD (ER associated degradation) pathway?

Answer 25

if the nascent misfolded glycoprotein cannot bypass the improper folding step of removing a glucose

Question 26

What does the ERAD pathway involve?

Answer 26

AAA ATPase p97

Question 27

What is AAA ATPase p97?

Answer 27

ER membrane protein utilizes ATP hydrolysis to pull misfolded proteins across ER membrane into cytosol (retrotranslocation)

Question 28

How is cystic fibrosis related to the ERAD pathway?

Answer 28

most patients possess mutant transporter protein (not properly glycosylation) - degraded by ERAD rather than properly targeted from ER to plasma membrane

Question 29

What happens once the redirected misfolded nascent glycoprotein is in the cytoplasm from N-linked glycosylation?

Answer 29

oligosaccharide chains removed and protein is poly-ubiquitinated

Question 30

what is poly-ubiquitination?

Answer 30

protein linked to chain of repeating poly ubiquitin units

Question 31

What is ubiquitin (UB)?

Answer 31

small protein involved in diverse cellular functions

Question 32

What is mono UB?

Answer 32

serves as signal for targeting membrane proteins into intraluminal vesicles of late endosomes multivesicular bodies

Question 33

What is poly UB?

Answer 33

serves as signal for ER protein degradation and for most other cellular proteins destined for normal turnover

Question 34

What is poly UB degraded by?

Answer 34

proteasome

Question 35

What is a proteasome?

Answer 35

barrel shaped multi-subunit protein degrading machine located in cytoplasm and nucleus

Question 36

What are the steps for the proteasome degradation of poly UB?

Answer 36

UB protein binds to cap of lid of the proteasome poly UB chain removed protein threaded into proteasome and degraded via proteolysis free AA are reused for new protein synth

Question 37

How can the ERAD pathway be overloaded?

Answer 37

if misfolded proteins accumulate in the ER to high levels - can lead to several diseases and results in ER stress

Question 38

What does ER stress signal?

Answer 38

unfolded protein response pathways (UPR)

Question 39

What are the three protein sensors that mediated UPR pathways?

Answer 39

Ire1 PERK ATF6

Question 40

What is a protein sensor?

Answer 40

ER integral membrane-spanning proteins

Question 41

What do PERK and ATF6 both have?

Answer 41

ER lumenal facing stress sensing domains which bind to molecular chaperones (BiP) in ER lumen

Question 42

What happens in the PERK and ATF6 pathways in stress and non-stress conditions?

Answer 42

non stress - PERK and ATF6 sensors inactive by binding to BiP in ER stress conditions UPR pathways are activated

Question 43

What are the steps to the PERK-mediated UPR pathway?

Answer 43

1. BiP released from PERK to aid in folding of accumulating ER proteins 2. PERK dimerizes and becomes active 3. cytoplasmic facing kinase domains of activated PERK dimer phosphorylate (inhibit) eIF2alpha (required for protein synthesis) 4. decrease in cellular protein synthesis and molecular chaperones (BiP) is available to focus on pre-existing proteins in the ER 5. ER stress alleviated, if not cell death

Question 44

What are the steps to the ATF6-mediated UPR pathway?

Answer 44

1. in ER stress, BiP released from ATF6 (BiP needed for folding accumulating misfolded ER proteins) 2. active ATF6 moves from ER to golgi via transport vesicles 3. at golgi, the cytoplasmic facing TF domain of ATF6 is cleaved off by a golgi-associated protease ATF6 TF domain targets to nucleus via an exposed NLS 4. in nucleus, ATF6 TF domain upregulates genes encoding key proteins involved in ER quality control (reticuloplasmins, ER export components which move proteins out of ER to golgi, and ERAD components) 5. ER stress is alleviated or cell death