Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Prinz, William A.; Spiess, Christoph; Ehrmann, Michael; Schierle, Clark; Beckwith, Jon

doi:10.1002/j.1460-2075.1996.tb00906.x

Cited by 62 publications

(66 citation statements)

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“…Presumably because the presence of a signal peptide is necessary but not sufficient to direct protein export, most of the hybrid proteins that we generated remained in the cytoplasm (data not shown). Indeed bona fide presecretory proteins may have a distinctive ability to remain loosely folded that greatly facilitates their export (41). Nevertheless, we found that about 25% of an MBP signal peptide-phosphoglycerate kinase fusion (MBP ss -PGK) was translocated across the IM.…”

Section: Figmentioning

confidence: 84%

Trigger Factor Retards Protein Export in Escherichia coli

Lee

Bernstein

2002

Journal of Biological Chemistry

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Trigger factor (TF) is a ribosome-associated protein that interacts with a wide variety of nascent polypeptides in Escherichia coli. Previous studies have indicated that TF cooperates with DnaK to facilitate protein folding, but the basis of this cooperation is unclear. In this study we monitored protein export in E. coli that lack or overproduce TF to obtain further insights into its function. Whereas inactivation of genes encoding most molecular chaperones (including dnaK) impairs protein export, inactivation of the TF gene accelerated protein export and suppressed the need for targeting factors to maintain the translocation competence of presecretory proteins. Furthermore, overproduction of TF (but not DnaK) markedly retarded protein export. Manipulation of TF levels produced similar effects on the export of a cytosolic enzyme fused to a signal peptide. The data strongly suggest that TF has a unique ability to sequester nascent polypeptides for a relatively prolonged period. Based on our results, we propose that TF and DnaK promote protein folding by distinct (but complementary) mechanisms.Protein biogenesis in Escherichia coli is aided by a diverse set of specialized factors that interact with nascent polypeptides chains. Several of these factors ("molecular chaperones") promote the folding of cytoplasmic proteins by binding promiscuously to exposed hydrophobic surfaces and preventing aggregation (reviewed in Ref.

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Section: Figmentioning

confidence: 84%

Trigger Factor Retards Protein Export in Escherichia coli

Lee

Bernstein

2002

Journal of Biological Chemistry

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“…The SecB/nascent chain interaction has also bcen shown to occur independently of the presence of a signal sequence [5]. Secondl~; translocation of some preproteins in which the signal scquences have been removed strictly requires SecB, even thot, gh they may not be SecB-dependent as a native preprotein [6,7]. The latter is likely a reflection of the targeting flmction of SecB, but again argues against the original kinetic partitioning model in which the retardation of folding by the signal sequence is an important element for recognition.…”

Section: Preprotein Targeting To the Membrane Secb Recognition Sites mentioning

confidence: 99%

“…The so-called pd (for protein localization) class of mutants, which are all isolated as suppressors of signal sequence mutations, have been found in SecA (prlD), SccY (pr/A), SecE (prlG) and more recently in SecG (prlH) (see references in [7,[36][37][38]). It has been proposed that the pr/suppressors function not by restoring the recognition of altered signal sequences but rather by preventing the rejection of defective preproteins from the export pathway [39].…”

Section: Signal Sequence Proofreading At the Initiation Of Translocationmentioning

confidence: 99%

The Sec system

Driessen

Fekkes

Wolk

1998

Current Opinion in Microbiology

159

111

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The Sec system Driessen, A.J.M.; Fekkes, P.; van der Wolk, J.P.W. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Proteins designated to be secreted by Escherichia coil are synthesized with an amino-terminal signal peptide and associate as nascent chains with the export-specific chaperone SecB. Translocation occurs at a multisubunit membrane-bound enzyme termed translocase, which consists of a peripheral preprotein-binding site and an ATPase domain termed SecA, a core heterotrimeric integral membrane protein complex with SecY, SecE and SecG as subunits, and an accessory integral membrane protein complex containing SecD and SecE Major new insights have been gained into the cascade of preprotein targeting events and the enzymatic mechanism of preprotein translocation. It has become clear that preproteins are translocated in a stepwise fashion involving large nucleotide-induced conformational changes of the molecular motor SecA that propels the translocation reaction. Addresses

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“…In addition, signal sequenceless TreA remained export competent, because it could be exported in prlA4 background (data not shown). prlA mutants are derivatives of secY allowing the export of signal sequenceless secretory proteins (18). To test whether treF could be actively expressed in the periplasm, we fused the signal sequence of TreA to the N terminus of TreF.…”

Section: Resultsmentioning

confidence: 99%

Determinants of Translocation and Folding of TreF, a Trehalase of Escherichia coli

Uhland¹,

Mondigler²,

Spiess³

et al. 2000

Journal of Biological Chemistry

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One isoform of trehalase, TreF, is present in the cytoplasm and a second, TreA, in the periplasm. To study the questions of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular compartment, we fused the signal sequence of periplasmic TreA to cytoplasmic TreF. Even though this TreF construct was exported efficiently to the periplasm, it was not active. It was insoluble and degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre ؉ revertants of periplasmic TreF. To further characterize periplasmic TreF, we used a genetic selection to isolate functional TreA-TreF hybrids, which were analyzed with respect to solubility and function. These data suggested that a domain located between residues 255 and 350 of TreF is sufficient to cause folding problems in the periplasm. In contrast to TreF, periplasmic TreA could fold into the active conformation in its nonnative cellular compartment, the cytoplasm, after removal of its signal sequence.Secretory and cytoplasmic proteins differ not only by the signal sequence but also in their folding properties. It is thought that the export competence of secretory proteins is the result of slow folding prior to export. Cytoplasmic proteins are believed to fold more rapidly and thus are not substrates of the cellular secretion apparatus. For a better understanding of the mechanism of translocation, the folding of cytoplasmic and secretory proteins needs to be characterized. Folding of polypeptides is determined by the primary amino acid sequence but also could be influenced by the particular properties of a cellular compartment. However, little is known about whether and how the cytoplasm and the periplasm specifically influence protein folding, i.e. whether there are other elements besides redox state and the presence/absence of ATP. To study these aspects, we used TreA and TreF, the two trehalases of Escherichia coli, as model proteins.Periplasmic TreA is synthesized as a precursor of 565 amino acids. The signal sequence of 30 amino acids is rather long.Mature TreA has a molecular mass of 58 kDa (1, 2). The K m of the purified enzyme is 0.8 mM, the V max is 66 mol of trehalose hydrolyzed/min/mg of protein, and the pH optimum is 5.5 (3). The expression of treA is independent of the presence of trehalose in the growth medium but is stimulated 10-fold at high osmolarity (1, 2). Also, treA is regulated by RpoS, the stationary phase sigma factor (4).Cytoplasmic TreF has 549 amino acids and a molecular mass of 64 kDa. The K m of the purified enzyme is 1.9 mM, the V max is 54 mol of trehalose hydrolyzed/min/mg of protein, and the pH optimum is 6.0. Like treA,

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Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Cited by 62 publications

References 50 publications

Trigger Factor Retards Protein Export in Escherichia coli

Trigger Factor Retards Protein Export in Escherichia coli

The Sec system

Determinants of Translocation and Folding of TreF, a Trehalase of Escherichia coli

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