Lecture 5

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

-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

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

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

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

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

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.

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)

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

KDEL receptors bind in a pH-dependent way

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.

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.

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