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Protein Processing in the Endoplasmic Reticulum
Andreas Herrlich MD PhDDepartment of Medicine,
Department of Cell Biology,
McDonnell Science Rm 863
Thank you to Phylis Hanson!
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Outline
• ER morphology• Protein folding• What happens when protein folding is
successful (Secretory pathway)– ER exit via COPII vesicles- Retrieval of ER residents from Golgi
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Endoplasmic reticulumThe ER is a compartment with a single continuous membrane and luminal space that can occupy up to 30% of cell volume
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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 (ERES, a.k.a. ribosome-free 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
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ER subdomains asdefined by proximityto other structures
Examples from cells expressingfluorescently taggedorganelle markers
Voeltz lab, UC Boulder
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Posttranslational modificationsProtein folding
Protein Processing and Quality Control in the Endoplasmic Reticulum
Unfolded Native
Unfolded protein response
ERAD: ER-associated degradationExit from the ER
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Protein Modifications and Folding in the ER
• Folding challenging in setting of ~400 mg/ml protein concentration
• “Hydrogel”• “Proteostasis”• In this unique milieu, proteins are continuously
challenged and “massaged” by a relentless folding machinery
Heterogeneous mixture of diverse proteins in different states of conformation, modification, oligomerization, and aggregation
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Protein Modifications and Folding in the ER
• Chaperones interact with unfolded proteins but not with properly folded proteins/protein complexes
• Compartimentalization of reactions within the lumen of the ER
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Role of classical chaperones• ER contains abundant Hsp70 and Hsp90 chaperones• Chaperones help other proteins acquire native
conformation, but do not form stable complexes
Hsp70s & Hsp90s bind exposed hydrophobic segments
BiP is main ER Hsp70, GRP94 is main ER Hsp90, interactions with clients are regulated by ATP status
Peptidyl-prolyl isomerases (PPIs) catalyse !cis–trans isomerisation of peptide bonds N-terminal to proline
Participation ofmany co-factorsin this regulation
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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
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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 (cargo capture “receptors”
Increases solubility
glucose
mannose
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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
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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
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0
500
1000
1500
2000
2500
0 0.2 0.4 0.6 0.8RNAse (mg)
cpm
Intact RNAse
Denatured RNAse
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 substrateMeasure incorporation of [14C] glucoseinto the oligosaccharide attached to �RNAse, compare native vs. denaturedRNAseResult: Only denatured RNAse is asubstrate.
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Fate of newly synthesized glycoproteins in the ER III
• Folding-defective proteins need to be degraded - transported out of the ER for degradation (red arrows)
• 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
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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
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VTC = Vesicular Tubular Cluster
TGN = Trans Golgi Network
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ER exit sites defined as sites of COPII vesicle formation
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Overview of COPII vesicle biogenesis
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Minimal COPII machinery
Five proteins added to liposomes or in vitro reactions form vesicles:
Sar1p, Sec23p, Sec24p, Sec13p, Sec31p
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How is cargo packaged into vesicles leaving the ER?Selective Nonselective
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Four core concepts of the early secretory pathway
• CARGO CAPTURE (selective)• BULK FLOW (nonselective)• RETENTION• RETRIEVAL
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ER!Vesicles!Vesicular Tubular clusters!cis Golgi
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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
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Diacidic motifs are common theme in efficiently secreted proteins
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
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COPII cargo binding site(s)
• 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)
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What about lumenal cargo?
Two possibilities: -bulk flow, with specific retention of ER resident proteins-receptor mediated exit via binding to secreted TM protein
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-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 COPIIvesicles per second
! Soluble proteins are efficiently secreted by bulk flow
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And what about large cargo?
Malhotra and ErlmannEMBO J 201130: 3475
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Cargo Capture + Bulk FlowLarge Cargo
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How is ER volume and content maintained?
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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 eukaryotes.
• Dilysine motif in C-terminal tail of receptor binds to COPI coat, lumenal domain binds HDEL/KDEL motif in pH dependent manner
http://www.ergito.com/lookup.jsp?expt=pelham
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Thank you!