“l23 protein functions as a chaperone docking site on the ribosome”

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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?