The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171.

Buchberger, Alexander; Valencia, Alfonso; McMacken, Roger; Sander, Chris; Bukau, Bernd

doi:10.1002/j.1460-2075.1994.tb06433.x

Cited by 121 publications

(128 citation statements)

References 39 publications

(29 reference statements)

Supporting

Mentioning

120

Contrasting

Order By: Relevance

“…DnaK and DnaJ were purified according to published protocols (21,22). GrpE carrying a C-terminally fused His 6 -tag was expressed from plasmid pUHE in an MC4100 ⌬dnaK strain (23) and purified via nickel-nitrilotriacetic acid-agarose and anion exchange chromatography (ResourceQ6 column, Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Hoffmann

Merz

Rutkowska

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

In prokaryotes, the ribosome-associated Trigger Factor is the first chaperone newly synthesized polypeptides encounter when they emerge from the ribosomal exit tunnel. The effects that Trigger Factor exerts on nascent polypeptides, however, remain unclear. Here we analyzed the potential of the Trigger Factor to shield nascent polypeptides at the ribosome. A set of arrested nascent polypeptides differing in origin, size, and folding status were synthesized in an Escherichia coli-based in vitro transcription/translation system and tested for sensitivity to degradation by the unspecific protease proteinase K. In the absence of Trigger Factor, nascent polypeptides exposed outside the ribosomal exit tunnel were rapidly degraded unless they were folded into a compact domain. The presence of Trigger Factor, as well as a Trigger Factor fragment lacking its peptidyl-prolyl isomerase domain, counteracted degradation of all unfolded nascent polypeptides tested. This protective function was specific for ribosome-tethered Trigger Factor, since neither non-ribosomal Trigger Factor nor the DnaK system, which cooperates with Trigger Factor in the folding process in vivo, revealed a comparable efficiency in protection. Furthermore, shielding by Trigger Factor was not restricted to short stretches of nascent chains but was evident for large, non-native nascent polypeptides exposing up to 41 kDa outside the ribosome. We suggest that Trigger Factor supports productive de novo folding by shielding nascent polypeptides on the ribosome thereby preventing untimely degradation or aggregation processes. This protected environment provided by Trigger Factor might be particularly important for large multidomain proteins to fold productively into their native states.Cells employ a large arsenal of molecular chaperones to promote the folding of newly synthesized proteins in the cytosol (1-3). At the forefront are chaperones, which transiently bind to ribosomes and thereby are optimally positioned to associate with emerging nascent polypeptides. In eubacteria, Trigger Factor (TF) 3 binds to ribosomes near the exit of the peptide tunnel through major contacts to L23 (4 -7) and interacts co-translationally with nascent polypeptides (8, 9). Downstream of TF, the DnaK-DnaJ-GrpE (referred to as KJE) and GroELGroES chaperone systems assist further folding steps to the native state (2, 10). Together these chaperones form a folding network of considerable robustness. Thus, the lack of TF can be compensated by activity of the KJE system, while simultaneous deletion of dnaK and the TF encoding tig gene results in synthetic lethality at Ն30°C and massive protein aggregation (11-13).TF is a three-domain protein of 48 kDa that adopts an unusual extended conformation (Fig. 1A) (5, 14). The N-terminal domain builds the "tail" of the molecule and harbors the "TF-signature motif," which mediates ribosome docking (4). A peptidyl-prolyl cis/trans isomerase (PPIase) domain with structural homology to FK506-binding proteins (9, 16) is located at the other end of ...

show abstract

Section: Methodsmentioning

confidence: 99%

Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Hoffmann

Merz

Rutkowska

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In contrast to DnaJ, the action of DnaK on the substrate protein requires continuous binding and release driven by ATP hydrolysis (45). To quantify the dynamics underlying this nonequilibrium process, we use a combination of manual and microfluidic mixing experiments, which allows us to monitor the process from milliseconds to hours.…”

Section: Dynamics Of Complex Formation From Microfluidicmentioning

confidence: 99%

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Kellner

Hofmann

Barducci

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

104

118

View full text Add to dashboard Cite

Molecular chaperones are an essential part of the machinery that avoids protein aggregation and misfolding in vivo. However, understanding the molecular basis of how chaperones prevent such undesirable interactions requires the conformational changes within substrate proteins to be probed during chaperone action. Here we use single-molecule fluorescence spectroscopy to investigate how the DnaJ-DnaK chaperone system alters the conformational distribution of the denatured substrate protein rhodanese. We find that in a first step the ATP-independent binding of DnaJ to denatured rhodanese results in a compact denatured ensemble of the substrate protein. The following ATP-dependent binding of multiple DnaK molecules, however, leads to a surprisingly large expansion of denatured rhodanese. Molecular simulations indicate that hard-core repulsion between the multiple DnaK molecules provides the underlying mechanism for disrupting even strong interactions within the substrate protein and preparing it for processing by downstream chaperone systems.Hsp70 | Hsp40 | Förster resonance energy transfer | protein folding | FRET

show abstract

“…A 70-kD contaminant (likely DnaK; Buchberger et al, 1994) was removed by anion exchange chromatography. The protein was exchanged into buffer B (50 mM Tris, 10 mM NaCl, and 0.02% NaN 3 ) using PD-10 desalting columns (GE Healthcare).…”

Section: Recombinant Protein Production and Purificationmentioning

confidence: 99%

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

et al. 2009

View full text Add to dashboard Cite

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.

show abstract

The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171.

Cited by 121 publications

References 39 publications

Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Contact Info

Product

Resources

About