“l23 protein functions as a chaperone docking site on the ribosome”
DESCRIPTION
“L23 Protein Functions as a Chaperone Docking Site on the Ribosome”. Kramer, G., et. al. (2002) Nature 419 171-174. Presented by Michael Evans Department of Chemistry and Biochemistry University of Notre Dame Notre Dame, IN 46616. Overview. Introduction to chaperones - PowerPoint PPT PresentationTRANSCRIPT
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“L23 Protein Functions as a Chaperone Docking Site
on the Ribosome”Kramer, G., et. al. (2002)
Nature 419 171-174
Presented by Michael EvansDepartment of Chemistry and BiochemistryUniversity of Notre DameNotre Dame, IN 46616
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Overview
• Introduction to chaperones• Experiments and Results• Conclusions• Future Work
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Chaperones and Folding
• Newly synthesized polypeptides must fold to native conformation in crowded environment of the cell
• Chaperones help many to avoid aggregation – Bind to exposed hydrophobic regions– PPIase activity– ATP dependent binding– Maintain conformational flexibility
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Chaperone Pathway in Bacteria
Hartl, F.U. and Hayer-Hartl, M. (2002) Science 295 1852-1858
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Trigger Factor (TF)
• First bacterial chaperone to see nascent polypeptide
• Has PPIase activity, but recognizes hydrophobic residues
• Function overlaps with DnaJ/DnaK chaperones
• N-terminal domain mediates binding to 50S subunit of ribosome
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Significance
• Explain coupling of synthesis to folding
• Eukaryotic parallels– No TF– Other chaperones interact with
ribosome– SRP study
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A Few Questions
• What part of TF is important for interaction with the ribosome?
• Which ribosomal protein(s) and/or RNA does TF interact with?
• Must TF bind ribosomes to interact with nascent chains?
• Is ribosomal association required for TF’s participation in protein folding?
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TF Signature
• Alignment of TF homologues revealed 17 conserved residues
• Completely conserved G-F-R-X-G-X-X-P motif--the TF signature
• TF signature located in unstructured region
• Could be surface-exposed and contribute to ribosome interaction
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TF Signature and Mutants
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TF Signature Mutants
• FRK/AAA: should show reduced association with ribosomes
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FRK/AAA Mutant Association with Ribosomes
• Incubated FRK/AAA with ribosomes from tig E. coli
• Ribosomes separated from unbound protein by centrifugation
• SDS-PAGE of pellet (ribosome) and supernatant (unbound protein)
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FRK/AAA Mutant Association with Ribosomes
•Increased amount of FRK/AAA in supernatant relative to wt TF incubated with ribosomes
S: SupernatantP: Ribosome Pellet
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TF Signature Mutants
• D42C: replace Asp with Cys to allow attachment of crosslinking reagent– BPIA is UV activatable– Attacks C-H bonds, so will react with ribosomal proteins
and RNA
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D42C Mutant Association and Crosslinking with Ribosomes• Couple TF D42C to BPIA• Incubate with tig ribosomes• Activate BPIA by UV irradiation • Separate ribosome-protein
complexes as before by centrifugation
• SDS-PAGE to resolve crosslinking products
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D42C Mutant Association and Crosslinking with Ribosomes
• Two products, 68 kDa and 75 kDa
• RNase A treatment does not affect mobility of products
• Trypsin digestion followed by ESI-MS to identify cross-linked proteins– 68 kDa: TF + L29
– 75kDa: TF + L23
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Interaction is Specific
• Add 2.5 M excess of either wt TF or FRK/AAA to compete with D42C-BPIA during crosslinking
• wt TF results in decrease of both crosslinking products
• FRK/AAA does not decrease yield of crosslinking products
• Crosslinking products are a result of a specific TF-ribosome interaction
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L23 and L29
• Both proteins of the large subunit• In direct contact with each other• Located next to the exit tunnel• Does TF associate directly with one
or both?
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L23 and L29 Deletion Mutants
Strategy: replace ORF with kanamycin resistance cassette
Adapted from Datsenko, K.A., and Wanner, B.L. (2000) Proc. Nat. Acad. Sci. 97 6640-6645
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L23 and L29 Deletion Mutants
• Two mutants produced: rpmC::kan, deletion of L29 gene rplW::kan, deletion of L23 gene
rpmC::kan grows, but slightly slower than wt
rplW::kan requires presence of pL23 for growth
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L23 and L29 Deletion Mutants
rplW::kan growth dependent on IPTG induction of pL23•L23 mutant is also viable
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L29 and TF Binding
• Purify ribosomes from rpmC::kan under high salt conditions
• Does TF remain bound to ribosomes without L29?
• Can TF rebind ribosomes without L29?
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TF Remains Associated to L29-Deficient Ribosomes
•SDS-PAGE of isolated ribosomes•Control is from rplW cells with wt L23 from plasmid•TF remains associated with L29-deficient ribosomes
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TF Can Rebind to L29-Deficient Ribosomes
•SDS-PAGE of ribosome-TF pellet and supernatant •Control is from rplW cells with wt L23 from plasmid•TF associates with L29-deficient ribosomes
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L23 Deletion and Mutants
• L29 is not required for TF binding, but what about L23?
rplW mutants are nonviable, but pL23 rescues
• What part of L23 is important for binding?
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L23 Region 1 and 2 Mutants• Criteria for interaction:
– residue is surface-exposed– Conserved among bacterial L23s
• Two regions identified
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L23 Region 1 and 2 Mutants
• Region 1: E18A, E18Q, VSE/AAA• Region 2: E52K, FEV/AAA• All mutant L23s complement rplW
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L23 Mutants and TF Binding
• Only region 1 mutants have effect on TF binding
• Does TF remain associated with ribosomes containing mutant L23?
• Can TF rebind ribosomes containing mutant L23?
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L23 Mutants and TF Binding
•SDS-PAGE of isolated ribosomes•Control is from rplW cells with wt L23 from plasmid•TF does not remain associated with mutant L23 ribosomes
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L23 Mutants and TF Binding
•SDS-PAGE of ribosome-TF pellet and supernatant •Control is from rplW cells with wt L23 from plasmid•Little TF binds to mutant L23 ribosomes
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L23 Mutants and TF Binding
•Less TF co-purifies with ribosomes under physiological salt concentrations•Mutant L23 levels are consistent with wt ribosomal proteins
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TF Interacts Directly with L23
• Create S-tagged L23-thioredoxin fusion (Trx-L23)
• Bind to S-tag column and apply TF or FRK/AAA
• Elute bound proteins
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TF Interacts Directly with L23
• TF binds L23, but FRK/AAA binding is weak
• TF and FRK/AAA have similar substrate binding properties
• L23-TF interaction is not mediated through nascent polypeptide
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TF • Nascent Polypeptide Interaction and L23
• Must TF bind L23 to interact with nascent polypeptide?
• Use in vitro transcription/translation (IVT) and crosslinking
• Produce 35S-labeled isocitrate dehydrogenase (ICDH) fragment
• Use crosslinker to probe for TF-ICDH interaction
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In Vitro Transcription/ Translation System
• Translation competent fraction from tig E. coli
• Purified ribosomes with wt L23, region 1 L23 mutants, or no L29
• Purified TF• Produce N-terminal fragment of
ICDH, an in vivo TF substrate
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Crosslinking
• Crosslinker is disuccinimidyl suberate (DSS)
• Homobifunctional• Spans 11.4 angstroms• Reacts with -amino groups of Lys to
give crosslink and N-hydroxy succinimide (NHS)
DSS
NHS
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Identifying Crosslink Results
• Immunoprecipitate crosslink product with anti-TF Ab
• IP and non-IP samples examined by elecrophoresis, autoradiography
• Control with no DSS
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L23 is Required for TF • ICDH Interaction
• wt L23 yields strong TF-ICDH crosslinks
• L23 mutants retard crosslinking
• Co-IP w/anti-TF Abs confirms identity
• Glu 18 mutants reduce TF-ICDH interaction
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TF-Ribosome Interaction and In Vivo Protein Folding
• Combine rplW::kan with dnaK• Compensate with plasmids for wt
or mutant L23• Examine growth and aggregation
at different temperatures
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TF-Ribosome Interaction and in vivo Protein Folding
• wt L23 compensates for deletion• L23 mutations lethal at 37ºC
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TF-Ribosome Interaction and in vivo Protein Folding
•Aggregates isolated from double mutants
•Aggregation increases with temperature
•VSE/AAA mutation is most severe
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The Big Picture
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Conclusions
• L23 is the TF docking site on the ribosome
• Glu 18 is critical for binding• Mutations in TF or L23 which
inhibit binding affect protein folding, growth
• L23 couples protein synthesis with chaperone-assisted folding
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Future Directions
• Why does TF form two crosslinks to nascent chains?
• What is the nature of the L23-TF binding interface?
• Does temp increase rate of aggregation or TF-L23 on-off rate?
• Role for eukaryotic L23 in recruiting chaperones?