1. Protein Processing in the Endoplasmic Reticulum
Phyllis Hanson
Cell Biology Dept., Cancer Res Bldg 4625

2. Outline ER morphology Protein folding
What happens when protein folding fails
ERAD
UPR
What happens when protein folding is successful
ER exit via COPII vesicles
- Retrieval of ER residents from Golgi

3. Endoplasmic reticulum

4. ] Subdomains of the ER Rough ER (mostly ER sheets or cisternae)
Protein translocation
Protein folding and oligomerization
Carbohydrate addition
ER degradation
Smooth ER (mostly ER tubules)
Lipid metabolism
Calcium release
Detoxification
ER exit sites (a.k.a. transitional ER) - export of proteins and lipids into the secretory pathway, marked by COPII coat
ER contact zones - transport of lipids, contact with other organelles
Nuclear envelope
Nuclear pores
Chromatin anchoring ]
About 1/3 of cellular protein
transits through the ER

5. ER subdomains as defined by proximity To other structures
Examples from
cells expressing
fluorescently tagged
organelle markers
Voeltz lab, UC Boulder

6. Protein Processing and Quality Control in the Endoplasmic Reticulum
Posttranslational modifications
Protein folding
Unfolded
Native
ERAD: ER-associated degradation
Unfolded protein response
Exit from the ER

7. Protein Modifications and Folding in the ER
Folding challenging in setting of ~400 mg/ml protein concentration
Folding facilitated and monitored by chaperones, both classical (Hsp70/Hsp90) and glycosylation dependent
Folded structure can be stabilized by disulfide bonds, facilitated by protein disulfide isomerases
Final folding can require assembly of multimeric protein complexes

8. Role of classical chaperones in ER protein folding
ER contains abundant Hsp70 and Hsp90 chaperones
Chaperones help other proteins acquire native conformation, but do not form stable complex
Hsp70s & Hsp90s bind exposed hydrophobic segments
Hsp70 in ER is BiP, interactions with client proteins regulated by ATP hydrolysis and exchange, large variety of cofactors control these
GRP94 is main ER Hsp90, also regulated by ATP status
PPIases–role of prolyl peptidyl cis-trans isomerases

9. Role of glycosylation dependent chaperones in ER folding
N-linked glycosylation:
Asn - X - Ser/Thr
Oligosaccharide addition
containing a total of
14 sugars
En bloc addition to protein;
subsequent trimming and
additions as protein progresses
through the secretory pathway;
five core residues are retained
in all glycoproteins

10. Fate of newly synthesized glycoproteins in the ER I
Increases solubility
Path when nascent protein folds efficiently (green arrows)
Players
OST = oligosaccharyl transferase
GI, GII = glucosidase I and II
Cnx/Crt = Calnexin and Calreticulin, lectin chaperones
ERp57 = oxidoreductase
ERMan1 = ER mannosidase 1
ERGIC53, ERGL, VIP36 = lectins that facilitate ER exit
glucose
mannose

11. Domain structure and interactions of calnexin
Model showing interaction of a folding
glycoprotein with calnexin and ERp57
Calnexin
binds other proteins
binds sugar
Williams, 2006 J Cell Sci 119:615

12. Fate of newly synthesized glycoproteins in the ER II
Path when nascent protein goes through folding intermediates (orange arrows)
Players
UGT1 (a.k.a. UGGT) = UDP-glucose–glycoprotein glucosyltransferase, recognizes “nearly native” proteins, acting as conformational sensor
Reglucosylated protein goes through Cnx/Crt cycle for another round
GII removes glucose to try again and pass QC of UGT1
BiP = hsc70 chaperone that recognizes exposed hydrophobic sequences on misfolded proteins

13. UDP-glucose glycoprotein glucosyltransferase (UGT1 a. k. a
UDP-glucose glycoprotein glucosyltransferase (UGT1 a.k.a. UGGT or GT) is an ER folding sensor
In vitro UGT1 reaction using
RNAse as glycoprotein substrate
Measure incorporation of [14C] glucose
into the oligosaccharide attached to RNAse, compare native vs. denatured
RNAse
Result: Only denatured RNAse is a
substrate.
Best substrates in vitro are “nearly folded glycoprotein intermediates”
not the native, compact structure or a terminally misfolded protein

14. Fate of newly synthesized glycoproteins in the ER III
Folding-defective proteins need to be degraded - transported out of the ER for degradation
How do proteins avoid futile cycles?
UGT1 does not recognize fatally misfolded proteins and won’t reglucosylate them for binding to Cnx/Crt
Resident mannosidases will trim mannose residues - protein can no longer be glucosylated and bind to Cnx/Crt
BiP binds hydrophobic regions
Mannosidase trimmed glycans recognized by OS9 associated with ubiquitination machinery
Leads to kinetic competition between folding and degradation of newly synthesized glycoproteins
Slow

16. Protein Processing and Quality Control in the Endoplasmic Reticulum
Posttranslational modifications
Protein folding
Unfolded
Native
Unfolded protein response
Exit from the ER
ERAD: ER-associated degradation

17. ERAD: ER-associated degradation

18. ERAD mechanism based on complete reconstitution
substrate
Stein et al. Cell (2014) 158:
Baldridge and Rapoport Cell (2016) 166:

19. What happens when ERAD isn’t enough and misfolded proteins accumulate?

20. Protein Processing and Quality Control in the Endoplasmic Reticulum
Posttranslational modifications
Protein folding
Unfolded
Native
Unfolded protein response
Exit from the ER
ERAD: ER-associated degradation

21. Unfolded Protein Response (UPR)
Intracellular signal transduction pathways that mediate communication between ER and nucleus
Activated by accumulation of unfolded proteins in the lumen of the ER
First characterized in yeast
Conserved and more complex in animals, with at least three pathways

22. The UPR in yeast Ire1=inositol-requiring protein-1,
ER-localized transmembrane kinase
and site specific endoribonuclease
Ire1 is maintained in inactive state by binding
to BiP. Removal of BiP (by binding to
misfolded proteins) leads to Ire1 activation
Ire1 activation triggers splicing
of intron in mRNA encoding Hac1, a
dedicated UPR transcriptional activator
Hac1 then binds to UPRE elements to
selectively upregulate gene expression
of targets that will help alleviate the
overload of misfolded proteins

23. Microarray analysis identified mRNAs for proteins up-regulated by the UPR
UPR induced in yeast by treatment with DTT or tunicamycin (Why??)
Travers et al. Cell (2000) 150:77-88

24. Unfolded Protein Response in Metazoans
Three branches
Cells respond to ER stress by:
Reducing the protein load that enters the ER
Transient
Decreased protein synthesis and translocation
Increase ER capacity to handle unfolded proteins
Longer term adaptation
Transcriptional activation of UPR target genes
Cell death
Induced if the first two mechanisms fail to restore homeostasis

25. UPR also has physiological roles
Rutkowski and Hegde, 2010

26. Misfolded proteins, ER stress, and disease
Cystic fibrosis transmembrane conductance regulator DF508 mutation is well studied example (among 100s known)
Protein could be functional as chloride channel at PM, but does not pass ER QC
Ameliorative strategies include use of chemical chaperones, efforts to modulate specific folding factors, and efforts to adjust overall “proteostasis”

27. UPR as Achilles heel in Multiple Myeloma?
UPR constitutively activated in setting of uncontrolled immunoglobulin secretion
May make cells particularly vulnerable to drugs that interfere with ER stress response, thereby increasing apoptosis
Proteasome inhibitors now in use, p97 inhibitors on the way
Others?

28. Protein Processing and Quality Control in the Endoplasmic Reticulum
Posttranslational modifications
Protein folding
Unfolded
Native
Unfolded protein response
Exit from the ER
ERAD: ER-associated degradation

30. ER exit sites defined as sites of COPII vesicle formation

31. Overview of COPII vesicle biogenesis

32. Minimal COPII machinery
Five proteins added to liposomes or in vitro reactions form vesicles:
Sar1p, Sec23p, Sec24p, Sec13p, Sec31p

34. How is cargo packaged into vesicles leaving the ER?

36. Live cell imaging of VSV-G transport
VSV-G ts045
mutant
tagged
with GFP
At 40°C, ts045 VSV-G is retained in the ER due to misfolding
Shift to 32°C - traffics to the plasma membrane

37. Specific amino acid signals mediate selective transport
Requirement of two acidic residues in the cytoplasmic tail of VSV-G for
efficient export from the ER. Nishimura & Balch, Science 1997

38. Diacidic motifs are common theme in efficiently secreted proteins
VSV-G TM-18aa -YTDIEMNRLGK
CFTR TM-212aa-YKDADLYLLD-287aaTM
GLUT4 TM-36aa -YLGPDEND
LDLR TM-17aa -YQKTTEDEVHICH-20aa
CI-M6PR TM-26aa -YSKVSKEEETDENE-127aa
E-cadherin TM-95aa -YDSLLVFDYEGSGS-42aa
EGFR TM-58aa -YKGLWIPEGEKVKIP-467aa
ASGPR H MTKEYQDLQHLDNEES-24aaTM
NGFR TM-65aa -YSSLPPAKREEVEKLLNG-74aa
TfR aa -YTRFSLARQVDGDNSHV-26aaTM

39. COPII cargo binding site(s)
(GAP)
Cargo binding sites recognize ER export signals in cytoplasmic domains of cargo
Best studied are the diacidic motifs in exiting membrane proteins, but there are others that bind to alternate sites in Sec24
(Cargo binding)

40. What about lumenal cargo?
Two possibilities:
-bulk flow, with specific retention of ER resident proteins
-receptor mediated exit via binding to secreted TM protein

41. -Measure secretion of soluble model protein
derived from Semliki Forest Virus capsid protein
-Protein folds rapidly, no need for chaperones
-Use pulse-chase analysis to follow newly
synthesized protein
-First molecule secreted 15 min after synthesis
-Rate constant of secretion is 1.2% per minute,
corresponding to bulk flow rate of 155 COPII
vesicles per second
Soluble proteins are efficiently secreted by bulk flow

42. And what about large cargo?
Malhotra and Erlmann
EMBO J 2011
30: 3475

44. How is ER volume and content maintained?

45. Retrograde traffic from Golgi to ER
Includes receptor-mediated mechanism for retrieving ER resident proteins
HDEL receptor identified in yeast - ERD2; multispanning transmembrane protein
KDEL receptor in higher euks.
Dilysine motif in C-terminal tail of receptor binds to COPI coat, lumenal domain binds HDEL/KDEL motif in pH dependent manner

46. Next lectures:
Tuesday:
Mechanism of membrane fusion
Secretory pathway organelles and trafficking
Thursday:
Endocytic pathways and organelles