Protein Processing in the Endoplasmic Reticulum

Phyllis Hanson

Cell Biology Dept., Cancer Res Bldg 4625

phanson22@wustl.edu

9/8/15

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

Endoplasmic reticulum

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 proteintransits through the ER

ER subdomains asdefined by proximityTo other structures

Examples from cells expressingfluorescently taggedorganelle markers

Voeltz lab, UC Boulder

Posttranslational modificationsProtein folding

Protein Processing and Quality Control in the Endoplasmic

Reticulum

Unfolded Native

Unfolded protein response

ERAD: ER-associated degradationExit from the ER

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

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

N-linked glycosylation:

Asn - X - Ser/Thr

Oligosaccharide additioncontaining 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

Role of glycosylation dependent chaperones in ER folding

Fate of newly synthesized glycoproteins in the ER I

• 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

Increases solubility

glucose

mannose

Domain structure and interactions of calnexin

binds sugar

binds other proteins

Williams, 2006 J Cell Sci 119:615

Model showing interaction of a foldingglycoprotein with calnexin and ERp57Calnexin

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

UDP-glucose glycoprotein glucosyltransferase (UGT1 a.k.a. UGGT or

GT) is an ER folding sensor

Best substrates in vitro are “nearly folded glycoprotein intermediates”not the native, compact structure or a terminally misfolded protein

In vitro UGT1 reaction usingRNAse as glycoprotein substrate

Measure incorporation of [14C] glucoseinto the oligosaccharide attached to RNAse, compare native vs. denaturedRNAse

Result: Only denatured RNAse is asubstrate.

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

Posttranslational modificationsProtein folding

Protein Processing and Quality Control in the Endoplasmic

Reticulum

Unfolded

Unfolded protein response

Native

ERAD: ER-associated degradation

Exit from the ER

ERAD: ER-associated degradation

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

Posttranslational modificationsProtein folding

Protein Processing and Quality Control in the Endoplasmic

Reticulum

Unfolded

Unfolded protein response

Native

ERAD: ER-associated degradation

Exit from the ER

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

The UPR in yeast

Ire1=inositol-requiring protein-1,ER-localized transmembrane kinaseand site specific endoribonuclease

Ire1 is maintained in inactive state by bindingto BiP. Removal of BiP (by binding tomisfolded 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 toselectively upregulate gene expressionof targets that will help alleviate theoverload of misfolded proteins

Microarray analysis identified mRNAs for proteins up-regulated by the UPR

Travers et al. Cell (2000) 150:77-88

UPR induced in yeast by treatment with DTT or tunicamycin (Why??)

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

UPR also has physiological roles

Rutkowski and Hegde, 2010

Misfolded proteins, ER stress, and disease

• Cystic fibrosis transmembrane conductance regulator F508 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”

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?

Posttranslational modificationsProtein folding

Protein Processing and Quality Control in the Endoplasmic

Reticulum

Unfolded

Unfolded protein response

Native

ERAD: ER-associated degradation

Exit from the ER

ER exit sites, a.k.a. transitional ER

Overview of budding at ER exit sites

A subset of SEC genes identified in the yeast Saccharomyces cerevisiae are the minimal

machinery for COPII vesicle buddingFive proteins added to liposomes or in vitro reactions form

vesicles:

Sar1p, Sec23p, Sec24p, Sec13p, Sec31p

Sec13/31p

Sec23/24p

Two layers of the COPII coat

Fath et al., Cell 129: 1326Stagg et al., Cell 134: 474

How is cargo packaged into vesicles leaving the ER?

Live cell imaging of VSV-G transport

At 40°C, ts045 VSV-G is retained in the ER due to misfoldingShift to 32°C - traffics to the plasma membrane

VSV-G ts045 mutanttagged with GFP

How are proteins concentrated for

secretion?Requirement of two acidic residues in the cytoplasmic tail of VSV-G for efficient export from the ER. Nishimura & Balch, Science 1997

Other transmembrane proteins with diacidic ER exit codes that direct incorporation into COPII

vesicles

VSV-G TM-18aa -YTDIEMNRLGKCFTR TM-212aa-YKDADLYLLD-287aaTMGLUT4 TM-36aa -YLGPDENDLDLR TM-17aa -YQKTTEDEVHICH-20aaCI-M6PR TM-26aa -YSKVSKEEETDENE-127aaE-cadherin TM-95aa -YDSLLVFDYEGSGS-42aaEGFR TM-58aa -YKGLWIPEGEKVKIP-467aaASGPR H1 MTKEYQDLQHLDNEES-24aaTMNGFR TM-65aa -YSSLPPAKREEVEKLLNG-74aaTfR -19aa -YTRFSLARQVDGDNSHV-26aaTM

Role of COPII coat in cargo selection

• 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

(GAP)

(Cargo binding)

What about lumenal cargo?

-Measure secretion of model protein, C-terminal domain of Semliki Forest VirusCapsid protein

-Chosen for its rapid, chaperone-independentFolding

-Use pulse-chase analysis to measure foldingand transport of newsly 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 COPIIvesicles per second

-Secretion is independent of expression level,and blocked by ATP depletion and BFA treatment,i.e. via classical secretory pathway

And what about large cargo?

Malhotra and ErlmannEMBO J 201130: 3475

Next lectures:

Thursday:

What happens when ER proteins do escape

Mechanism of membrane fusion

Secretory pathway organelles and trafficking

Tuesday:

Endocytic pathways and organelles

Reading• Lodish, 7th edition. Relevant

sections of chapters 13 and 14 OR• Pollard, 2nd edition, Chapter 20, pp.

355-360

• REVIEW TO READ: “Cleaning up: ER-associated degradation to the rescue”. JL Brodsky, Cell 151: 1163-1167

• Others of interest on website