Lecture 5

Question 1

Specificity of Protein Transport

Answer 1

Inclusion: only proteins interacting with coat is packaged
Exclusion: proteins with signals are not packed

Question 2

Bulk Flow Model

Answer 2

-Secretion is the default pathway and signals would keep it from being swept away
-example that proves this wrong is beta-lactamase which has a signal for secretion

Question 3

How was Bulk Flow tested?

Answer 3

Tripeptide Experiment: tracked N-linked oligosaccharide labeled with radioactive tripeptide, they then measure by recovering tripeptide
this was unfair since they did not take into account the folding of proteins

Question 4

Pulse Labeling

Answer 4

pulse and chase models
-biosynthetic labeling
-labeling of surface or endocytic compartments
-detecting changes use Immunoprecipitation for tracking

Question 5

Immunoprecipitation
How are they quantified?

Answer 5

isolating proteins using antigen antibody interaction
-subcellular fractionation - movement between compartments
-molecular weight shift - glycosidase
-fluorescence

Question 6

KDEL

Answer 6

• c-terminal signal for ER localization
• necessary ( if deleted, protein gets secreted)
  -sufficient( if added to a secreted protein, it stays in ER)

Question 7

Retrieval vs Retention

Answer 7

Retrieval is like an open prison, they can go anywhere but is still brought back
Retention is like a closed prison, they are anchored by a signal.

Question 8

Retrieval Model

Answer 8

Hypo: There’s a receptor that binds to KDEL proteins and retrieve them back
Evidence: There are KDEL proteins that seem to have modified Glc-NAc indicating that they’ve been to Golgi and encountered an enzyme and was retrieved back to ER (Cathepsin D)

Question 9

If BiP is not anchored in ER but is still free to move in ER, does it disprove the retention hypothesis?

Answer 9

BiP is remains in ER because COPII vesicles excludes them as they have low affinity for ligand-bound KDEL receptors at pH 7.2

Question 10

KDEL Receptors

Answer 10

KDEL receptors bind in a pH-dependent way

Question 11

How KDEL receptors bring back runaway ER proteins back to ER?

Answer 11

Receptors that are not in ER are protonated with acidic pH and readily binds with KDEL sequences and COPI vesicles. When ER proteins are in the COPI, they go back to ER. ER proteins are released to ER as receptor becomes deprotonated with ER ph 7 which now readily binds to COPII vesicles.

Question 12

Other ER retention signal sequences

Answer 12

KKxx and KxKxx are arginine-based sequences that are important for transmembrane proteins. These motifs are recognized by the coatemer of COPI.
Type I proteins have the motif on the C-terminal on the cytosolic side.
Type II proteins have the motif on the N-terminal on the cytosolic side with K/HDEL on the C-terminal sequence on the ER lumen.

Question 13

Evidence supporting receptor-mediated transport

Answer 13

Secretory proteins are concentrated during ER export in COPII vesicles compared to ER.