Outline

• Biosynthetic Pathway (ER)
• S-S, cotranslational, remodeled by PDI
• Glycosylation in ER
• Quality Control: lectin chaperones
• UPR
• Start w/ flow diagram, mention vesicle formation & maturation, retrograde flow, and compartment specific processing.

List steps for full protein synthesis:

• S-S
• folding, Hsc70 & quality control lectin chaperones
• multimerization
• initial glycosylation (ER)
• quality control (UPR), ER export
• final glycosylation (Golgi subcompartments)
• proteolytic activation
• targeting to destination
• So, important point #1: proper folding required for ER export: ie the first 4 steps.

• function is to stabilize tertiary & quaternary structure
• important:
• cytoplasm = reducing
• determined by GSH (50:1 over GSSG, due to NADPH)
• GSH= glu-cys-gly
• NADPH + H+ + GSSG -> NADP+ + 2GSH
• this reaction not present in ER lumen
• lumen= oxidizing
• S-S form sequentially cotranslationally
• rearranged by PDI if necessary, see diagram

Glycosylation: chains of saccharides added to lumenal domains

• function: important for folding, quality control, targeting, protection, and interactions
• 2 types:
• O-glycosylation: short chains occurs on ser or thr (hydroxyl->O)
• N-glycosylation: long chains occurs at Asp in Asp-X-Ser/Thr (Asp->N)
• the saccaride units are:
• man, glc, gal, fuc, GlcNAc, (GalNAc), NANA
• precursors made in cytosol as nucleotide diphosphates
• antiporter brings them into lumen
• added to chain by transferases with liberation of nucleotide diphosphate
• O added to protein one at a time
• N assembled as lipid precursor and added to protein en bloc
• important step #1: transfer to N from dolichol
• see diagram of man9
• step #2: used as quality control mech. for folding
• 1man and all 3glc removed
• if unfolded-> add 1 glc back
• transiently bind lectin chaperone to prevent aggregation
• upon release glc then removed again and cycle repeated until folded
• see diagram of cycle

Unfolded protein response (UPR)

• misfolded proteins are re-exported, ubiquinated and degraded by proteosome
• accumulation of unfolded proteins (stress) triggers UPR: see diagram
• ER(inner membrane) TM protein IRE1 detects(dimerized) unfolded protein
• nuclear domain of IRE1 contains endonuclease that cleaves Hac1 pre-mRNA
• cleaved Hac1 ligated by tRNA ligase
• Hac1 mRNA exported and translated
• Hac1 transcription factor imported & activates chaperone genes