Polypeptide Flux through Bacterial Hsp70

Teter, Sarah; Houry, Walid; Áng, D. D.; Tradler, Thomas; Rockabrand, David M.; Fischer, Gunter; Blum, Paul H.; Georgopoulos, Costa; Hartl, F. Ulrich

doi:10.1016/s0092-8674(00)80787-4

Cited by 387 publications

(175 citation statements)

References 49 publications

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“…This function may not necessarily be carried out by a "classic" Hsp70. For example, the ribosome-bound peptidyl-cis-trans isomerase, trigger factor is a prime candidate for a nascent chain chaperone in E. coli, which like Ssb can be cross-linked to short nascent chains (Lill et al, 1988;Deuerling et al, 1999;Teter et al, 1999).…”

Section: Ssb a Fungal-specific Hsp70mentioning

confidence: 99%

“…Until an entire domain is synthesized, the translating protein likely remains unfolded, thereby exposing hydrophobic patches to the crowded environment outside the ribosome. Chaperones are thought to protect newly synthesized chains from aggregation by binding to these exposed hydrophobic patches until the protein is able to fold correctly (Horwich et al, 1993;Hartl, 1996;Deuerling et al, 1999;Teter et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s

Pfund¹,

Huang²,

Lopez-Hoyo

et al. 2001

MBoC

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Ssbs of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, which can be cross-linked to nascent polypeptide chains. Because Ssbs are members of a divergent subclass of Hsp70s found thus far only in fungi, we asked if the structural requirements for in vivo function were similar to those of “classic” Hsp70s. An intact peptide-binding domain is essential and an alteration of a conserved residue in the peptide-binding cleft (V442) affects function. However, Ssb tolerates a number of alterations in the peptide-binding cleft, revealing a high degree of flexibility in its functional requirements. Because binding of Ssb to peptide substrates in vitro was undetectable, we assessed the importance of substrate binding using the chimera BAB, in which the peptide binding domain of Ssb is exchanged for the analogous domain of the more “classical” Hsp70, Ssa. BAB, which binds peptide substrates in vitro, can substitute for Ssb in vivo. Alteration of a residue in the peptide-binding cleft of BAB creates a protein with a reduced affinity for peptide and altered ribosome binding that is unable to substitute for Ssb in vivo. These results indicate that Ssb's ability to bind unfolded polypeptides is likely critical for its function. This binding accounts, in part, for its stable interaction with translating ribosomes, even although it has a low affinity for peptides that detectably bind to other Hsp70s in vitro. These unusual properties may allow Ssb to function efficiently as a chaperone for ribosome-bound nascent chains.

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Section: Ssb a Fungal-specific Hsp70mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s

Pfund¹,

Huang²,

Lopez-Hoyo

et al. 2001

MBoC

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“…The GrpE protein mediates transfer of a substrate protein from the DnaK system to GroEL/GroES (Bukau & Horwich, 1998). The DnaK system may also cooperate with ClpB93/ClpB79, HtpG Mogk et al, 1999;Thomas & Baneyx, 2000;Schlieker et al, 2002), the small heat-shock proteins IbpA/IbpB (Thomas & Baneyx, 1998;Veinger et al, 1998;Shearstone & Baneyx, 1999) or trigger factor, a peptidyl-prolyl isomerase having a chaperone activity (Hesterkamp et al, 1996;Teter et al, 1999). A possible role for GrpE in the interaction of the DnaK/DnaJ and ClpB systems emerges from the experiments of Zolkiewski (1999) on suppression of luciferase aggregation in vitro.…”

Section: Introductionmentioning

confidence: 99%

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells

et al. 2004

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Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.

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“…In vivo, trigger factor has been found to be non-essential. Lethality results only when trigger factor depletion occurs in combination with depletion of DnaK, the bacterial Hsp70 homolog [7,8].…”

Section: Introductionmentioning

confidence: 99%

Is Ffh required for export of secretory proteins?

Kim¹,

Rusch²,

Luirink³

et al. 2001

FEBS Letters

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In Escherichia coli, protein export from the cytoplasm may occur via the signal recognition particle (SRP)-dependent pathway or the Sec-dependent pathway. Membrane proteins utilize the SRP-dependent route, whereas many secretory proteins use the cytoplasmic Sec machinery. To examine the possibility that signal peptide hydrophobicity governs which targeting route is utilized, we used a series of PhoA signal sequence mutants which vary only by incremental hydrophobicity changes. We show that depletion of SRP, but not trigger factor, affects all the mutants examined. These results suggest secretory proteins with a variety of signal sequences, as well as membrane proteins, require SRP for export. ß

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Polypeptide Flux through Bacterial Hsp70

Cited by 387 publications

References 49 publications

Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s

Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells

Is Ffh required for export of secretory proteins?

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