Vesicular Transport

Question 1

Where is N-linked glycosylation of membrane proteins initiated in the ER?

Answer 1

lumen

Question 2

Describe N-linked glycosylation of membrane proteins in the ER.

Answer 2

1. A branched oligosaccharide is transferred from a lipid moiety to an asparagine residue that resides in the sequence Asn-X-Thr or Asn-X-Ser (where X is any amino acid)
2. N-linked sugar modifications can direct sorting to different membrane compartments in cells, and can also be involved in cell-cell recognition

Core sugar is further modified during transport through the Golgi.

Question 3

Formation of disulfide bonds in the ER is limited to what kinds of proteins?

Answer 3

secretory and membrane proteins (occurs during or soon after synthesis)

Question 4

On membrane proteins in the ER, are disulfide bonds typically in the lumen or the cytosol? Why?

Answer 4

Lumen. The reducing environment of the cytosol prevents disulfide bond formation

Question 5

What enzyme catalyzes the breakage and reformation of disulfide bonds in the lumen of the ER allowing the protein to achieve its thermodynamically most stable conformation?

Answer 5

protein disulfide isomerase

Question 6

What ensures proteins are properly folded prior to exiting the ER?

Answer 6

Chaperones (Proper folding of proteins is required for transport out of the ER. )

Question 7

Oligomeric proteins of the secretory pathway, such as antibody molecules with their 2 heavy and 2 light chains, are assembled where?

Answer 7

in the ER. Transport out of the ER does not take place until assembly has occurred.

Question 8

What happens to mis-folded proteins in the ER?

Answer 8

They are reverse-transported out of the ER through the translocation channel. In the cytosol, they are deglycosylated, ubiquitylated and subsequently degraded

Question 9

What are mis-folded proteins degraded by?

Answer 9

Proteasome

Question 10

What is stimulated by the accumulation of mis-folded proteins in the ER?

Answer 10

The synthesis of ER chaperones. Misfiled proteins binds to receptors in the ER lumen. These receptors send a transcription factor to the nucleus to stimulate the chaperone gene be transcribed and translated and transported into the ER to aiding in folding

Question 11

What is emphysema and what is it caused by?

Answer 11

a disease in which lung tissue is destroyed by the uncontrolled activity of elastase. The major genetic cause appears to be due to a mutation in α1-anti-trypsin (an inhibitor of elastase) that results in improper folding and the transport of the mis-folded protein out of the ER for degradation by the proteasome.

Question 12

What is the main function of Alpha-1 anti-trypsin?

Answer 12

Inhibits elastase (If no inhibition of elastase there is damage to the lung causing emphysema)

Question 13

What is the common mutation of Alpha-1 anti-trypsin that can lead to emphysema?

Answer 13

AA sequence

Question 14

What causes Cystic Fibrosis?

Answer 14

F508del mutation of the CFTR protein, oligosaccharide side chains are not processed properly. As a consequence, the protein mis-folds and is degraded.

Question 15

Movement of proteins from the ER to the Golgi and then to lysosomes, the plasma membrane or secretory vesicles occurs how?

Answer 15

via transport vesicles (this process involves coat proteins, SNARES, and RABs)

Question 16

What directs the Budding of Vesicles from the Different Membrane Compartments ofthe Secretory Pathway?

Answer 16

coat proteins (and form basket-like complexes which help deform the membrane before it is pinched off to form the vesicle)

Question 17

What are the three types of coat proteins for secretory vesicles budding from the ER?

Answer 17

Clathrin, COPI, and COPII

Question 18

A clathrin coat protein with clathrin and GGA adaptor regulate transport to and from where?

Answer 18

to lysosome from Golgi apparatus (via endosomes)

Question 19

A clathrin coat protein with clathrin and adaptin 1 regulate transport to and from where?

Answer 19

to plasma membrane from Golgi apparatus (clathrin dependent exocytosis)

Question 20

A clathrin coat protein with clathrin and adaptin 2 regulate transport to and from where?

Answer 20

to endosomes from plasma membrane (aka involved in clathrin dependent endocytosis)

Question 21

A COPI coat protein guides vesicles to and from where?

Answer 21

vesicles moving within the Golgi apparatus in an anterograde fashion

AND

on vesicles formed in the Golgi that are destined for the ER membrane in a retrograde fashion

Question 22

A COPII coat protein guides vesicles to and from where?

Answer 22

vesicles formed in the ER that are destined for the cis-Golgi (anterograde transport)

Question 23

What induces budding in a clathrin coated vesicle?

Answer 23

the clathrin lattice formed from triskelions

Question 24

What do adaptin molecules do in clathrin coat vesicles?

Answer 24

bind specific transmembrane proteins and cargo receptors to prepare for budding (membrane deformation and budding is mechanistically the same regardless of the coat)

Question 25

What enzyme pinches off a clathrin-coated protein during budding of a vesicle?

Answer 25

Dynamin (a GTPase). Other coats use similar mechanisms. If dynamin was lost, budding would not proceed

Question 26

After budding, do vesicles with clathrin and adaptin 2 maintain their coat proteins long?

Answer 26

No. Soon after pinching off the vesicles lose their protein coat. It uncoats to uncover other targeting structures on the vesicle.

Question 27

Make sure to be able to visualize the different morphologies of cathrin, COPI, and COPII coat proteins

Answer 27

Reference slide 14 of lecture

Question 28

Is COPI involved in anterograde or retrograde transport?

Answer 28

Both.
Anterograde= through the Golgi (cis to trans).
Retrograde= Golgi back to ER

Question 29

Is COPII involved in anterograde or retrograde transport?

Answer 29

Only Anterograde (from ER to Golgi)

Question 30

Targeting of vesicles to the correct membrane compartment involves rabs, tethers, and SNAREs. How does this work?

Answer 30

1. RAB and v(vesicle)-snare bind to the vesicle and RAB binds to a tethering protein on the target membrane in a process called ‘Tethering’
2. v-snare on the vesicle bind specific t(target membrane)-SNAREs found on the target membrane compartment in a process called ‘Docking’
3. The very tight inter-wrapping of the v-SNARE and the t-SNARE pulls the membranes closer together and promotes membrane fusion. (aka ‘Fusion’ step)

NOTE: the coat protein is gone by this point

NOTE: this is process involved in acetylcholine release at the synaptic cleft

Question 31

Proteins leaving the ER in anterograde fashion are coated by what?

Answer 31

COPII

Question 32

Retrieval of ER cargo receptors and soluble ER resident proteins (aka lumen proteins) that were mis-sorted to the cis-Golgi occurs via what coat protein?

Answer 32

COPI (retrograde fashion)

Question 33

How does retrieval of transmembrane ER proteins from the Golgi occur?

Answer 33

via binding of a cytosolic di-lysine sequence directly to the COPI coat that guides it back .

Question 34

How does the retrieval of lumenal ER proteins from the Golgi apparatus work?

Answer 34

This process is mediated by an ER retention signal on the lumenal ER protein. ER retention signal is a four amino acid sorting signal (KDEL, Lys-Asp-Glu-Leu) found at the carboxy terminus. The KDEL sequence binds to a KDEL receptor in the cis Golgi membrane and results in the selective retrieval of these ER proteins from the Golgi.

Question 35

Do proteins that exit the ER with COPII coats enter the Golgi apparatus on the cis or trans face?

Answer 35

Cis (they then receive additional sugar modifications as they transition to the trans-face)

Question 36

Info on: visualizing the sorting of proteins through the secretory pathway using real-time imaging techniques

Answer 36

1. ***The use of proteins that have been fused to GFP (green fluorescent protein) has enabled the visualization of the individual steps in membrane protein sorting.
2. The molecular machinery that mediates the individual steps in sorting have often been defined through the use of temperature sensitive mutations.
   a. Temperature sensitive mutant have wild type activity at the permissive temperature and are defective at the restrictive temperature. Many of these studies were done in yeast because of the ease with which mutants can be generated (most mutant proteins identified in this way have mammalian homologues).

Question 37

Proteins are targeted to the plasma membrane by a default pathway from the trans-network of the Golgi apparatus.

Answer 37

This constitutive secretory pathway of exocytosis occurs independent of any known stimulus.

This class of proteins has no specific targeting sequence for exiting the trans Golgi network.

Question 38

Proteins exit the Golgi from the trans Golgi network and can undergo delivery to which three membrane compartments?

Answer 38

1. The plasma membrane
2. Secretory vesicles (hormones)
3. Lysosomes

Question 39

What is Familial Hyperproinsulinemia?

Answer 39

An asymptomatic condition in which there are elevated levels of proinsulin due to a defect in the proteolytic processing of insulin

Question 40

Regulated secretion or exocytosis is common in proteins exiting the trans-golgi and destined for the plasma membrane. When do these proteins fuse with their respective target membranes?

Answer 40

The contents of these vesicles are stored in special secretory vesicles and are released only upon neurotransmitter or hormonal stimulation of the cell.

Question 41

Formation of a mature secretory vesicle involves which coat-protein?

Answer 41

Clathrin. Proteins are often proteolytically processed in secretory vesicles during formation of a mature vesicle (ex: proinsulin to insulin)

Mature secretory vesicles are densely packed with protein in a clathrin dependent process, More so than in the golgi

Question 42

Is the lumen of the ER a reducing or oxidizing environment?

Answer 42

oxidizing

Question 43

What environment conditions might cause mis-folding of proteins in the ER?

Answer 43

heat shock or cold shock

Question 44

Patients who experience coldness in their feet from this drug that prevents microtubule disassembly?

Answer 44

plecitaxel

Question 45

What are the SNAREs and RAB involved with acetylcholine docking?

Answer 45

VAMP (v-snare) and syntacin (t-snare) and RAB3A