Protein Processing in the Endoplasmic Reticulum Phyllis Hanson Cell Biology Dept., Cancer Res Bldg /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 protein transits through the ER

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

Posttranslational modifications Protein folding Protein Processing and Quality Control in the Endoplasmic Reticulum UnfoldedNative Unfolded protein response ERAD: ER-associated degradation Exit 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 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 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 folding glycoprotein with calnexin and ERp57 Calnexin

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 using RNAse as glycoprotein substrate Measure incorporation of [ 14 C] glucose into the oligosaccharide attached to RNAse, compare native vs. denatured RNAse Result: Only denatured RNAse is a substrate.

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 modifications Protein 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 modifications Protein 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 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

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 modifications Protein 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 budding Five 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: 1326 Stagg 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 misfolding Shift to 32°C - traffics to the plasma membrane VSV-G ts045 mutant tagged 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-GTM-18aa -YTDIEMNRLGK CFTRTM-212aa-YKDADLYLLD-287aaTM GLUT4TM-36aa -YLGPDEND LDLRTM-17aa -YQKTTEDEVHICH-20aa CI-M6PRTM-26aa -YSKVSKEEETDENE-127aa E-cadherinTM-95aa -YDSLLVFDYEGSGS-42aa EGFRTM-58aa -YKGLWIPEGEKVKIP-467aa ASGPR H1 MTKEYQDLQHLDNEES-24aaTM NGFRTM-65aa -YSSLPPAKREEVEKLLNG-74aa TfR -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 Virus Capsid protein -Chosen for its rapid, chaperone-independent Folding -Use pulse-chase analysis to measure folding and 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 COPII vesicles 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 Erlmann EMBO J : 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, 7 th edition. Relevant sections of chapters 13 and 14 OR Pollard, 2nd edition, Chapter 20, pp REVIEW TO READ: “Cleaning up: ER- associated degradation to the rescue”. JL Brodsky, Cell 151: Others of interest on website