Lecture 1 - Exam 4: Protein Sorting and Vesicular Trafficking

Question 1

The synthesis of proteins from the endoplasmic reticulum is destined for?

Answer 1

Destined for the Golgi, endosomes, lysosomes, plasma membrane, and/or secretory vessels.

Question 2

The synthesized proteins from the ER is destined for the Golgi, endosomes, lysosomes, plasma membrane, and/or secretory vessels. What determines protein localization?

Answer 2

Signal sequence.

Question 3

Where does the synthesis of lipids occur?

Answer 3

Smooth ER.

Question 4

What kind of posttranslational modifications happen and where do they happen?

Answer 4

Glycosylation and attachment of lipids are modifications that occur in the ER.

Question 5

What are the three different orientations of membrane proteins?

Answer 5

There two types that span the membrane once, but differ in whether the carboxyl (C) or amino (N) terminus is on the cytosolic side.
There is another orientation that has multiple membrane-spanning regions.

Question 6

Describe the insertion of a membrane protein with a cleavable signal sequence.

Answer 6

Some transmembrane proteins have a normal amino terminal signa; sequence. The signal is cleaved by the signal peptidase as the polypeptide chain crosses the membrane.

Question 7

Describe translocation of a membrane protein with a cleavable signal sequence that is inserted into the ER.

Answer 7

Translocation is inhibited by the membrane spanning domain (~25 hydrophobic a.a.’s). This opens the translocon and allows the hydrophobic transmembrane (TM) domain to move laterally into the membrane.

Question 8

Describe the insertion of membrane proteins with internal transmembrane sequences.

Answer 8

Other proteins are inserted into the ER by internal TM sequences that are recognized by SRP, but not cleaved by the peptidase.
These internal sequences are then brought to the translocon and the ribosome pushes the rest of the a.a. chain into the lumen of the ER.

Question 9

The orientation of the membrane proteins depends on?
What is it driven by?

Answer 9

Depends on the amino acids immediately flanking the TM domain = positively charged amino acids stay on the cytosolic side of the translocon.
This is driven by negatively charged residues near the cytosolic side of the translocon.

Question 10

Describe the insertion of membrane proteins that span the membrane multiple times.

Answer 10

The protein is inserted into the ER by internal TM sequences that are recognized by SRP, but not cleaved by the peptidase (like membrane proteins with internal transmembrane sequences). Again, these internal sequences are brought into the translocon and the ribosome pushes the rest of the a.a. chain into the lumen. With the same protein still intact, a second transmembrane sequence is inserted into the translocon (not recognition by SRP here, only in the beginning). The ribosome pushes the rest of the a.a. chain into the CYTOSOL. A third transmembrane sequence is inserted into the translocon and the rest of the a.a. chain is pushed into the lumen.

Question 11

Describe posttranslational insertion of a protein with a C-terminal transmembrane sequence.

Answer 11

C-terminus is not recognized by SRP because the C-terminal sequence does not exit the ribosome until translation is complete. TRC40 brings them to a different TM receptor in the ER, GET1-GET2.

Question 12

What happens during posttranslational translocation of proteins in the ER?

Answer 12

The Hsp70 chaperones or BiPs are thought to bind the protein as it leaves the translocon and then mediates protein folding in the ER.

Question 13

After the Hsp70 chaperones or BiPs bind the protein as it leaves the translocon, the protein is folded in the ER (mediated by the chaperone). What happens to correctly folded proteins? What about incorrectly folded proteins?

Answer 13

Correctly folded proteins can go on to the Golgi. Incorrectly folded proteins are targeted for degradation.

Question 14

Does the ER have a reductive environment or an oxidative environment? What does this type of environment allow?

Answer 14

An oxidative environment. This allows disulfide bonds (S-S) to form via the enzyme protein disulfide isomerase (PDI).

Question 15

Does the cytosol have a reductive environment or an oxidative environment?

Answer 15

Reductive environment.

Question 16

What is O-glycosylation? What is N-glycosylation?

Answer 16

O-glycosylation: The attachment of a sugar to the hydroxyl of serine (Ser, S), threonine (Thr, T).
N-glycosylation: The attachment of a sugar to the amide group of asparagine (Asn, N).

Question 17

What are the predominant sugars found in glycoproteins?

Answer 17

N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc)

Question 18

Describe the structure of N-glycosylation (Asparagine).

Answer 18

Core group of two N-acetylglucosamines (GlcNAc) and three mannoses (complex sugars).

Question 19

Describe the structure of O-glycosylation (Serine, Threonine).

Answer 19

Core group of one N-acetylgalactosamine (GalNAc).

Question 20

Remember only specific Asn (N) residues are glycosylated. What are those residues?

Answer 20

Asn – X – Ser
or
Asn – X – Thr

X is any amino acid except proline.

Question 21

Addition of GPI anchors:
What are glycosylphosphatidylinositol (GPI) anchors?

Answer 21

GPI anchors are assembled in the ER. They contain two fatty acid chains linked to an inositol head group and an oligosaccharide portion consisting of mannose and glucosamine residues.

Question 22

Addition of GPI anchors:
GPI anchors have an oligosaccharide portion consisting of mannose and glucosamine residues. What are these added to?

Answer 22

These are added to polypeptides anchored in the membrane by a C-terminal membrane-spanning region.**

Question 23

Addition of GPI anchors:
GPI anchors have an oligosaccharide portion consisting of mannose and glucosamine residues that are added to polypeptides anchored in the membrane by a C-terminal membrane-spanning region. What happens after that?

Answer 23

The membrane-spanning region is cleaved and the new carboxy terminus is joined to the NH2 group of ethanolamine, leaving the protein attached to the membrane by the GPI anchor.

Question 24

Describe the Quality control in the ER (Calnexin-Calreticulin pathway).

Answer 24

1. Glycoprotein exits the translocon, two glucose residues are removed.
2. Calnexin (or calreticulin*) recognizes the glycoprotein and remove another glucose.
3. Protein folding sensor. It will monitor hydrophobic regions.
   -If there are no problems:
   Protein will pass the quality control and will exit the ER.
   -If there are some problems:
   The folding sensor will add back a glucose residue, allowing the protein to bind back to Calnexin to attempt correcting the folding.
   -Several issues and the protein stayed too long:
   It will be recognized by EDEM1, which removes mannose residues. This prevents the protein to bind calnexin and target protein to a pathway of degradation (proteasome).

Question 25

If an excess of unfolded proteins accumulates what happens?

Answer 25

A signaling pathway called the unfolded protein response (UPR) is activated. ER-associated degradation (ERAD).

Question 26

If there’s an excess of unfolded proteins accumulates, the unfolded protein response is activated. What does this lead to?

Answer 26

It leads to expansion of the ER and production of more chaperone proteins.

Question 27

The Unfolded Protein Response:
If protein folding can’t be adjusted to a normal level, what happens?

Answer 27

The cell undergoes programmed cell death (apoptosis).

Question 28

What are the 3 receptors that the UPR activates in the ER?

Answer 28

IRE1, ATF6, and PERK

Question 29

What does the activation of IRE1, ATF6, and PERK cause?

Answer 29

Activation of these receptors causes transcription of genes encoding chaperones lipid synthesis enzymes, and ERAD proteins.

Question 30

Draw the UPR receptor actions

Answer 30

Draw them so you can remember! Slide 58

Question 31

With the PERK receptor, what happens to eIF2?

Answer 31

eIF2 is phosphorylated by PERK and therefore inhibits eIF2. When active, eIF2 brings the tRNA to the ribosome. So, that will not happen. Translation is then “dampened down” and trying to prevent too many folded proteins.