1 Protein Processing in the Endoplasmic Reticulum Phyllis Hanson Cell Biology Dept., Cancer Res Bldg 4625 [email protected] 9/19/11 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 – Bulk flow vs. facilitated forward transport
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1!
Protein Processing in the Endoplasmic Reticulum �
Phyllis Hanson�
Cell Biology Dept., Cancer Res Bldg 4625 �
9/19/11 �
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�– Bulk flow vs. facilitated forward transport�
2!
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�
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Posttranslational modifications!Protein folding!
Protein Processing and Quality Control in the Endoplasmic Reticulum
Unfolded! Native!
Unfolded protein response!
ERAD: ER-associated degradation!!!
Exit from the ER!!!
Protein Modifications and Folding in the ER �
�• Addition and processing of carbohydrates�• Proper folding is both facilitated and
monitored by chaperone interactions�• Formation of disulfide bonds�• Assembly into multimeric proteins�
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Consensus sequence: ��Asn - X - Ser/Thr �
Oligosaccharide addition�containing a total of �14 sugars �
N-Linked Glycosylation �
En bloc addition to protein; �subsequent trimming and �additions as protein progresses �through the secretory pathway;�five core residues are retained �in all glycoproteins �
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�
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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 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 �
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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 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.��
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�
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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�"�
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ERAD: ER-associated degradation
The Ubiquitin-Proteasome System (UPS) is essential for ERAD, degrades proteins in the cytoplasm �
E1 = ubiquitin activating enzyme�E2 = ubiquitin conjugating enz.�E3 = ubiquitin ligase � "Multiple family members to � "recognize specific substrates,
"specific ER ubiquitin ligases "dedicated to ERAD �
�
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Vembar & Brodsy NRMCB 2008 9:944�
What happens when ERAD isn’t enough �and misfolded proteins accumulate?�
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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 which have one branch �
• Conserved and more complex in animals, with three primary branches��
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The UPR in yeast
Ire1=inositol-requiring protein-1,�ER-localized transmembrane kinase�and site specific endoribonuclease��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 UPR elements to�upregulate their expression�
Ire1p is a transmembrane serine-threonine kinase,oriented with the amino terminus (N) in the ER lumenand the carboxyl terminus in the cytosol. Whenunfolded proteins accumulate in the ER, Ire1poligomerizes, trans-autophosphorylates via thecytosolic kinase domain (K) and activates theendonuclease in the tail domain (T). Theendonuclease Ire1p cuts HAC1 mRNA at two sites,removing a nonclassical intron; the exons are rejoinedby Rig1p (tRNA ligase). HAC1u (uninduced) is nottranslated owing to the presence of the intron, andHacpu is not produced (brackets). After Ire1p-mediated splicing, HACIi mRNA is efficientlytranslated into Hac1pi, a transcriptional activator thatupregulates expression of UPR target genes afterbinding to the unfolded protein response element(UPRE) in the promoters of genes encoding ER-resident chaperones and other proteins. Patil and Walter 2001!
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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�
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UPR also has physiological roles
Rutkowski and Hegde, 2010�
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Misfolded proteins, ER stress, and disease�
• Cystic fibrosis transmembrane conductance regulator ΔF508 mutation is well studied example (among 100s known)�
• Protein could be functional as chloride channel at PM, but does not pass ER QC �
• Strategies to ameliorate problem include use of chemical chaperones, efforts to modulate specific folding factors, and efforts to adjust overall “proteostasis”�
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�"�
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ER exit sites, a.k.a. transitional ER
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Overview of budding at ER exit sites
A subset of SEC genes identified in the yeast Saccharomyces cerevisiae define the minimal
machinery for COPII vesicle budding from the ER !
Five proteins added to liposomes or in vitro reactions form vesicles:��! !Sar1p, Sec23p, Sec24p, Sec13p, Sec31p!
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Sec13/31p�
Sec23/24p!
Two layers of the COPII coat!
Fath et al., Cell 129: 1326 �Stagg et al., Cell 134: 474!
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How is cargo packaged into vesicles leaving the ER?�
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Live cell imaging of VSV-G transport�using GFP fusions �
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�
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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 -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 H1 ! MTKEYQDLQHLDNEES-24aaTM!NGFR ! !TM-65aa -YSSLPPAKREEVEKLLNG-74aa!TfR ! ! -19aa -YTRFSLARQVDGDNSHV-26aaTM!
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Role of COPII coat in cargo selection
• Cargo binding sites recognize ER export signals in cytoplasmic domains of cargo�
• Best studied are diacidic motifs in exiting membrane proteins, but there are others that bind to alternate sites in Sec24�
(GAP)!
(Cargo binding)!
What about lumenal cargo? �
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-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 2011�30: 3475 �
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Next lectures:��Thursday:��What happens when ER proteins do escape��Mechanism of membrane fusion��Secretory pathway organelles and trafficking���Tuesday: ��Endocytic pathways and organelles����
Textbook Reading�• Lodish, 6th edition. Section 13.3 pp.
549-556 or�• Pollard, 2nd edition, Chapter 20, pp.
355-360 or �• Alberts, 5th edition. Chapter 12, pp.
723-745 ��
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