Quiz questions

The classic experiments of George Palade and his colleagues first demonstrated the sequence of organelles that constitute the secretory pathway. It is:

Which organelle receives proteins and lipids from the endoplasmic reticulum, modifies them, and then dispatches them to other destinations in the cell?

Which ONE of the following proteins does NOT enter the secretory pathway?

Which of the following is true about posttranslational modifications?

Proteins are properly folded with the assistance of

Which of the following are used to assist in protein folding?

I.  calnexin II. calreticulin III. Hsp70 IV. disulfide bonds

Which of these diseases does not directly involve protein aggregation?

Which disease can be caused by too much ER stress?

The unfolded protein response (UPR) operates at which levels of regulation? I. transcription initiation II. mRNA decay III. translational initiation IV. protein degradation

Secretory pathway

Palade's experiment (pulse chase)

1st order view: ![[Pasted image 20260304091830.png]]

Golgi is soul of secretory pathway

Cis Golgi -> Medial Golgi

Complementation assay Basically combined mutant cells expressing a protein but lacked enzyme that incorporates H-GlcNAc with Medial Golgi form wild type cells.

Membrane traffic scheme

Cargo selection Vesicle budding Takes coat proteins around vesicle, scission from donor membrane Vesicle transport Uncoating in cytosol Vesicle docking Tethering by tethering factor. Grabbed by t-snare proteins from acceptor membrane Vesicle fusion Exocytosis with acceptor membrane

Protein Quality Control

Job of ER Protein folding Disulfide bond formation Glycosylation (N-linked) Proteolytic cleavage Formation of multimeric complexes Send things onward to Golgi Lipid synthesis (cytoplasmic face) Regulation of cholesterol synthesis

Protein folding

Chaperones in ER (BIP) Bip pulls proteins through membrane (as being translated, BIP can attatch) Disulfide isomerase folds protein until lowest energy state Disulfide bonds created by cystein

Intermolecular disulfide bonds (oligomers) in proinsulin

Bonds between insulin, when you have a lot ER stress response

Cystic Fibrosis

A site of phenyalanine deletion = makes protein slow to fold = too long to fold = gets degraded

Glycosylation and trimming in ER lumen (Glycosylation as quality control)

Unfolded protein glucose binded Calnexin (chaperone) chops of terminal glucose N-linked oligosaccharide shows its properly folded If incomplete fold, goes to glucosyl transferase. Uses UDP-glucose -> UDP to properly fold If cant be folded at all, exit from ER, destroyed Then exit from ER

ER stress response

Reduce mRNA = Less stuff going to ER Reduce Misfolded proteins = Less stuff Reduce translation = less stuff Increase chaperones = help with folding Bigger ER volume (Up regulate lipid synthesis) = more room to help Reduce Cell growth = less stress Reduce transcription = les stuff

IRE1 and PERK Key signalers of the UPR

PERK

phosphorylates a translation initiation factor = reduce translation = good for ER stress Down regulates eIF2 alpha Slows translation of most mRNAs PERK upregulates XPB1, but need IRE1 to splice it

IRE1

Kinase/endonuclease that splices its target XPB1

1. Senses unfolded proteins, forms oligomers, targets phosphorylation on mRNA sitting on trans Locon
2. Usually no splicing at XPB1 intron, but IRE1 can, makes XBP1 proteins = good for ER stress

ER associated degradation (ERAD)

Targets terminally misfolded proteins for degradation Gets proteins back out of ER through reverse translocation process into cytoplasm. Goes to proteasome in cytoplasm, degraded

How could IRE1 and PERK sense misfolded proteins? Look for hydrophobic parts exposed Disulfide badly folded bonds

Type 2 Diabetes

Example of ER stress (A bunch of insulin folded up).