ER-GOLGI TRAFFICKING AND ENDOCYTOSIS

Question 1

Cotranslation / translocation steps

Answer 1

1. The N’ end of the peptide emerging from the ribosome contains a signal sequence targeting it to the RER
2. Signal recognition particle (SRP) binds to the signal sequence and stops translocation
3. SRP binds to an SRP receptor associated with a closed channel (translocon) on the RER membrane, mediating GTP binding
4. GTP binds the SRP & SRP receptor and hydrolysis opens translocon and kicks off the SRP to be recycled
   
   1. Translation restarts w/o SRP, translating the peptide through the open translocon
5. Signal peptidase cleaves off the signal peptide
6. For a non-integral soluble protein, the entire peptide is formed inside the ER lumen
7. Ribosome falls off mRNA and RER, then dissociates into 40S and 60S
8. Translocon closes, peptide folds

Question 2

What proteins are translocated through the ER membrane and into the ER lumen/matrix?

What proteins are inserted directly into the ER membrane?

Answer 2

soluble matrix proteins

integral proteins

Question 3

What’s the first modification that occurs, either within the ER or Golgi?

What does it help with?

Answer 3

Glycosylation: enzymatic addition carbohydrates/glycans

• protein folding/targeting
• protein stability
• cell-to-cell contact via lectins (carbohydrate-binding proteins)
• antigens (e.g. ABO blood typing)

Question 4

Non-enzymatic mechanism of adding carbohydrates

Answer 4

Glycation

Question 5

O-linked glycosylation

Answer 5

Addition of individual carbohydrates to -OH groups on Ser or Thr by glycosyl transferases

Ex) Addition of carbohydrates to RBCs to generate ABO blood groups

Question 6

N-linked glycosylation

Answer 6

1. Oligosaccharyl transferase adds glycans to proteins as a preformed oligosaccharide complex to asparagine (Asn) in an Asn-X-(Ser/Thr) consensus sequence
   
   1. Occurs in the ER while the peptide is translocated through the ER membrane
2. Glycosidases further process the complex
3. Final product signals that the glycosylated protein is ready for transport from the ER to the Golgi, where it may be further processed

Question 7

The oligosaccharide precursor of N-linked glycosylation is initially synthesized where?

Where is it finalized?

Answer 7

Initially built upon a dolichol phosphate in the cytosol on the outersurface of the ER.

Later finalized in the ER lumen

Question 8

Activated nucleotide sugar donors, ___ & ___, add the sugars to dolichol phosphate sequentially until the oligosaccharide precursor for N-linked glycosylation is complete

Answer 8

UDP-sugar or GDP-sugar

Question 9

Tunicamycin

Answer 9

inhibitory analog of UDP-gluNAc, which is the first nucleotide-sugar substrate in formation of the oligosaccharide precursor of N-linked glycosylation.

–> stops N-linked glycosylation

–> prevents folding, stability, targeting, and secretion

Question 10

What enzyme transfers the oligosaccharide precursor complex from the dolichol phosphate to the Asn residue of the consensus sequence?

Answer 10

Oligosaccharyl transferase

Question 11

What kind of proteins tend to have disulfide bonds?

Answer 11

Secreted proteins

&

Proteins on the extracellular leaflet of the plasma membrane

Question 12

Disulfide bonds aid in

Answer 12

protein folding by providing strong covalent structure & stability

Question 13

You woul dnever find disulfide bonds in proteins synthesized from free ribosomes because

Answer 13

Disulfide bonds form in the ER lumen only under oxidative conditions, and as the protein is translocated through the ER membrane.

Question 14

PDI

Answer 14

Protein disulfide isomerase transfers oxidized disulfide bonds from its Cys to reduce dprotein substrates –> forms and rearranges disulfide bonds

Question 15

What does A-1AT do?

Answer 15

Inhibits trypsin and elastase to prevent excessive elastase (from neutrophils) damaging the lungs.

Question 16

Alpha-1 antitrypsin (A-1AT) deficiency

Answer 16

• Point mutation in the A-1AT protein prevents proper folding in the ER of hepatocytes
  
  • –> Crystalline aggregates can’t be secreted and build up, damaging the liver and impairing secretion of other liver proteins
    
    • Liver diseases (jaundice, cirrhosis) in children
  • –> Excessive elastase activity damages lung
    
    • Familial emphysema in adults

Question 17

A-1AT deficiency is treated by

Answer 17

Immunizations to prevent lung infection

Avoid smoking and lung irritants

A-1AT replacement therapy

Bronchodilators

Inhaled steroids to open airways

O2 administration

Liver transplant (most common)

Question 18

After translcoation into the ER and processing in the ER, proteins are ultimately transported to function in what 3 possible places?

Answer 18

Golgi

Endosomes/lysosomes

Plasma membrane

Question 19

Anterograde vs Retrograde transport

Answer 19

• Anterograde: vesicular movement from
  
  • ER to the cis-Golgi
  • Trans-golgi to endosomes/lysosomes or plasma membrane
• Retrograde: vesicles return proteins to previous compartments in the process
  
  • Ex) cis-Golgi to the ER, trans to medial

Question 20

Cisternal maturation

Answer 20

New cis-Golgi forms when ER-to-golgi anterograde vesicles bud and fuse with it –> moves previous cis-Golgi up to the medial Golgi position –> moves previous medial Golgi up to trans-Golgi

• Proteins get processed and/or sorted into vesicles
• Cisternae mature through retrograde vesicles transporting enzymes
  
  • Ex) When ER vesicle fuses to form a new cis-Golgi, retrograde vesicles transport needed ER enzymes back tothe ER

Question 21

What anterograde vesicles move from the ER to the cis-Golgi?

What is the associated GTPase?

Answer 21

COPII vesicles

Sar1

Question 22

What are the retrograde vesicles?

What is their GTPase?

Answer 22

COPI

ARF

Question 23

Clathrin-coated vesicles transport from where to where?

What’s their coat protein?

Associated proteins

Their GTPase?

Answer 23

• TGN -> endosomes or lysosomes
• Plasma membrane -> endosomes, lysosomes, other cell-specific structures (endocytosis)

Coat protein: clathrin

Associate adaptor protein: AP1, AP2, AP3

GTPase: ARF

Question 24

Describe ER to cis-Golgi anterograde transport for a soluble cargo protein

Answer 24

1. Soluble cargo binds to an ER membrane-bound receptor
2. COPII coat protein binds to the receptor and causes formation of a curved COPII vesicle
3. Vesicle pinches off and de-coats, exposing v-SNAREs
4. The v-SNARE binds to the t-SNAREs on the target membrane, the cis-Golgi –> vesicle fusion
5. Soluble cargo is now bound to its receptor, bound in the cis-Golgi membrane

Question 25

How would the ER to cis-golgi anterograde transport be different for an integral membrane cargo protein (as opposed to a soluble one)?

Answer 25

• COPII coat protein binds to the cytosolic domain of the integral membrane cargo
  
  • _{(instead of binding to the receptor for soluble cargo)}
• The end product places the integral cargo in the cis-Golgi membrane
  
  • _{(instead of bound to a receptor)}

Question 26

Sar1 and ARF are GTPases - what are their 2 important funcitons?

Answer 26

vesicle formation

&

de-coating

Question 27

Describe the GTPase function of Sar1

Answer 27

• GTP-bound Sar1 inserts its hydrophobic tail into the ER membrane to interact with COPII coat proteins
  
  • –> binds coat protein to the vesicle membrane, thus recruiting cargo, and promoting curvature for vesicle formation
• Hydrolysis –>GDP-bound Sar1 retracts its tail –> de-coating

Question 28

After the ER, the preformed N-linked oligosaccharide complex goes to the cis-Golgi by anterograde vesicle transport.

What kind of processing could happen here?

Answer 28

High mannose glycosylation in the cis-Golgi

Complex glycosylation in the trans-Golgi

Modifications of the high mannose structure can target the protein to the lysosome.

Question 29

From the TGN, where can proteins be targeted?

Answer 29

• Previous trans-Golgi (retrograde transport)
• Lysosomes
• Late endosomes
• Constitutive or regulated secretion

_{Remember: The transport vesicles <u>usually</u> have a clathrin coat protein (exceptions), adapter protein, and ARF}

Question 30

What’s the function of the AP?

Answer 30

It binds a cytosolic domain sorting signal on the cargo or cargo receptors AND the coat protein

–> gets cargo into the correct vesicle

Question 31

Transport from TGN to trans-Golgi would use..

Answer 31

COPI (retrograde)

ARF

_{clathrin is not the coat protein}

Question 32

Transport from TGN to the lysosome would use

what AP?

what GTPase?

what signal?

Answer 32

_{Maybe clathrin}

AP3

ARF

Cargo signal: Mannose6-phosphate

Question 33

Transport from the TGN to the endosome involves

what coat protein?

what ap?

what GTPase?

What about from the plasma membrane to the endosome (receptor-mediated endocytosis)?

Answer 33

• TGN to endosome
  
  • Clathrin, AP1, ARF
• Receptor-mediated endocytosis
  
  • AP2 & ARF

Question 34

Secretion/exocytosis can be __ or __

Answer 34

constitutive or regulated

_{Regulated secretion involves aggregation}

Question 35

Child with coarse facial features, craniofacial abnormalities, psychomotor retardation, and regression of developmental psychomotor milestones

Answer 35

I-cell disease: defect in lysosomal transport

Question 36

In transporting to the lysosome,

1. M6P-containing lysosomal proteins are packaged into AP3 vesicles in the TGN –> bud off
2. Vesicle de-coats, recycling the clathrin and AP3
3. Uncoated vesicle fuses w/ lysosome (or late endosome that matures into lysosome)

What happens to the receptor?

Answer 36

• It is recycled back to the TGN or goes to the plasma mambrane
  
  • Goes to plasma membrane in case a hydrolase is accidentally constitutively secreted –> receptor can bind it in the ECM and bring it back into the late endosome/lysosome

Question 37

If lysosomal proteins aren’t tagged with the M6P signal, like in I-cell disease, what happens?

Answer 37

Constitutive secretion of ALL lysosomal proteins (not just the hydrolases) and none are ever returned to the lysosome via receptor-mediated endocytosis

–> lysosomal storage disease

Question 38

Creation of the M6P sortign signal

Answer 38

1. GlcNAc phosphotransferase uses UDP-GlcNAc as a substrate to add GluNAc-phosphate to one of the Mannose sugars.
2. Phosphodiesterase removes Glu-NAc, leaving the Mannose 6-phosphate sorting signal

Question 39

I-cell disease / Mucolipidosis II

Answer 39

• Defective GlcNAc phosphotransferase –> can’t make M6P so all proteins destined for the lysosome are mis-targeted and secreted from the cell, causing a buildup of inclusion bodies (carbs & lipids) in the lysosome
  *

Question 40

ML-I

ML-II

ML-IIII

Answer 40

ML-I: sialidase deficiency

ML-II/ I-cell disease: defective GlcNAc phosphotransferase

ML-IIII/Pseudo-Hurler polydystrophy: less severe deficiency of GlcNAc phosphotransferase

Question 41

What would you seein a patient with pseudo-Hurler polydystrophy or I-cell disease?

How could you differentiate?

Answer 41

• Cells filled with dense inclusion bodies
• High level of lysosomal enzymes in patient’s serum and body fluids

Pseudo-hurler polydystrophy ocurs later in life and the patient surives past the first decade

Question 42

How are I-cell diseae and psuedo-Hurler polydystrophy different from other lysosomal storge disorders?

Answer 42

Both result from a targeting defect, not a single enzyme defect –> ALL lysosomal enzymes are affected

Question 43

In both phagocytosis/pinocytosis and receptor-mediated endocytosis. endosomes are formed and ..

Answer 43

acidified and enriched with acid hydrolases –> becomes late endosomes, and ultimately lysosomes

Question 44

Phagocytosis involves evagination of the plasma membranae to engulf a foreign particle and internalize it in…

Answer 44

membrane-enclosed phagosomes

Question 45

Autophagy

Answer 45

removal of old and worn-out organelles into autophagosomes for digestion

• Cell may digest its organelles as a stress response
• Plays a role in immunity and infection, destroying intracellular pathogens
• Can result from inflammation and is increase in neural injury/degeneration

Question 46

Steps of receptor-mediated endocytosis for soluble cargo protein (or the receptor itself)

Answer 46

1. Integral receptor binds a cargo protein
2. Either the receptor or the cargo binds to AP2
3. AP2 interacts with Clathrin
4. ARF decoats clathrin, creating an uncoated vesicle (Early endosome)
5. Early endosome gains other acid hyrolases & ATP-dependent proton pump via vesicular trafficking from TGN –> late endosome
6. Late endosome becomes a lysosome as pump keeps lowering pH
7. Ligand-receptor complexes dissociated and the receptors are recycled to the PM or degraded in the lysosome

Question 47

The LDL particle transports cholesterol. Its hydrophobic core contains cholesteryl-esters.

What on its outer surface serves as the ligand for RME?

How does it get digested by lysosomes?

Answer 47

ApoB

The LDL receptor on a cell surface binds ApoB for RME.

In the acidic endosome or lysosome where the pH is lower, the receptor becomes positively charged, decreasing its affinity for ApoB –> LDL particle is released into lysosome for digestion

Question 48

The LDL receptor is bound in _____

At neutral pH, it will bind ApoB on the LDL particle, causing internalization of the ligand-receptor complex into a ____

the vesicle de-coats, creating an early endosome that acidifies into a late endosome.

The acidic pH promotes release of LDL into the lysosme, while the LDL receptor is ___

Answer 48

The LDL receptor is bound in a clathrin-coated pit to AP2

At neutral pH, it will bind ApoB on the LDL particle, causing internalization of the ligand-receptor complex into a clathrin-coated endocytic vesicle

The vesicle de-coats, creating an early endosome that acidifies into a late endosome. The acidic pH promotes release of LDL into the lysosome, while the LDL receptor is forms into another vesicle that recycles back to the PM

Question 49

Familialhypercholesteremia occurs due a

Answer 49

mutation in the LDL receptor (absence, defective, premature degradation, inability to sort) or in the ApoB ligand –> LDL builds up in blood –> plaques

Homozygous is worse than heterozygous

Question 50

Fe3+ + apotransferrin = ferrotransferrin

Describe iron transport

Answer 50

1. Ferrotransferrin binds to the transferrin receptor on cell surface
2. Receptor-mediated endocyotsis
3. Vesicle uncoats and acidification of the late endosome releases the iron from ferrotransferrin
4. Apotransferrin binds transferrin receptor in the acidic pH of the late endosome
5. Apotransferrin-transferrin is recycled back to the PM, where neutral pH causes them to dissociate and apotransferrin is released

Question 51

What happens when a receptor on the plasma membrane or in the Golgi needs to be degraded, NOT recycled?

Answer 51

The membrane and proteins invaginate to become vesicular bodies within the late endosome. Because the vesicle is completely within the lysosomal matrix, it can be degraded by acid hydrolases.

Question 52

Formation of vesicular bodies

Answer 52

1. Hrs proteins and proteins required for degradation get monoubiquitinated
2. Monoubiquitinated Hrs interacts with ESCRT to induce invagination of endosomal multivesicular bodies that are then degraded
3. ESCRT disassembled and recycles

_{this process is ued in the budding of HIV particles}

Question 53

HIV budding

Answer 53

1. HIV Gag gets monoubiquitinated to serve as a scaffold for ESCRT assembly on the plasma membrane–> evagination into the extracellula rmatrix

Gag is part of the HIv envelope

Question 54

Describe the role of acid hydrolases for the lysosomal degradation of cellular macromolecules.

Answer 54

Proteases, glycosidases, nucleases, lipases, sulfatases and phosphatases in lysosomes are collectively referred to as acid hydrolases, in that they possess maximal activity in an acidic environment.

Inactive in the cytosolic ph

Question 55

In receptor-mediated endocytosis, does the clathrin coat directly bind the cargo/receptor to be internalized?

Answer 55

NO

Instead, the integral receptor (or cargo) is bound through interactions between its cytosolic domain and the AP2 protein. AP2 interacts with Clathrin

Question 56

apotransferrin has a high affinity for the transferrin receptor under what kind of pH?

Answer 56

low pH, like in the lysosome when it binds the transferrin receptor to be recycled back to the PM together

Back at the neutral pH of the extracellular matrix, apotransferrin has a low affinity for the transferrin receptor, and apotransferrin is released.