The β subunit polypeptide of Vibrio harveyi luciferase determines light emission at 42° C

Escher, Alan; O’Kane, Dennis J.; Szalay, Aladar A.

doi:10.1007/bf00280295

Cited by 13 publications

(9 citation statements)

References 27 publications

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“…Twelve amino acid substitutions in this protein enhance its ability to fold in bacterial cells at high temperatures (31). In wild-type yeast cells, substantial activity was detected with this protein at 34°C (Fig.…”

Section: Resultsmentioning

confidence: 93%

In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone

Nathan

Vos

Lindquist

1997

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

341

264

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In the highly concentrated environment of the cell, polypeptide chains are prone to aggregation during synthesis (as nascent chains await the emergence of the remainder of their folding domain), translocation, assembly, and exposure to stresses that cause previously folded proteins to unfold. A large and diverse group of proteins, known as chaperones, transiently associate with such folding intermediates to prevent aggregation, but in many cases the specific functions of individual chaperones are still not clear. In vivo, Hsp90 (heat shock protein 90) plays a role in the maturation of components of signal transduction pathways but also exhibits chaperone activity with diverse proteins in vitro, suggesting a more general function. We used a unique temperature-sensitive mutant of Hsp90 in Saccharomyces cerevisiae, which rapidly and completely loses activity on shift to high temperatures, to examine the breadth of Hsp90 functions in vivo. The data suggest that Hsp90 is not required for the de novo folding of most proteins, but it is required for a specific subset of proteins that have greater difficulty reaching their native conformations. Under conditions of stress, Hsp90 does not generally protect proteins from thermal inactivation but does enhance the rate at which a heat-damaged protein is reactivated. Thus, although Hsp90 is one of the most abundant chaperones in the cell, its in vivo functions are highly restricted.

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Section: Resultsmentioning

confidence: 93%

In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone

Nathan

Vos

Lindquist

1997

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

341

264

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“…The luciferase is a heterodimer composed of two subunits, ␣ with an M r of 40,000 to 45,000 and ␤ with an M r of 35,000 to 40,000. We compared the small and large subunits of the PII A synthase with the ␣ and ␤ subunits of luciferases from different bioluminescent bacteria, such as Vibrio harveyi (16) and Vibrio fischeri (18), and found only a weak identity between them. The highest scores obtained (17 to 19% identity) were always between SnaB and the ␣ or ␤ luciferase subunits, in the Nterminal regions.…”

Section: Discussionmentioning

confidence: 99%

Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase

Blanc¹,

Lagneaux²,

Didier³

et al. 1995

J Bacteriol

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. 177:5199-5205, 1995); these enzymes are PII A synthase, a heterodimer composed of the SnaA and SnaB proteins, which catalyzes the oxidation of PII B to PII A , and the NADH:riboflavin 5-phosphate oxidoreductase (hereafter called FMN reductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes designed from limited peptide sequence information of the purified proteins, the corresponding genes were cloned from a genomic library of S. pristinaespiralis. SnaA and SnaB showed no significant similarity with proteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulation of PII B . Complementation of a S. pristinaespiralis PII A ؊ PII B ؉ mutant with the snaA and snaB genes, cloned in a low-copy-number plasmid, partially restored production of PII A . The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN reductases but was 39% identical with the product of the actVB gene of the actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furthermore, an S. coelicolor A(3)2 mutant blocked in this step was successfully complemented by the snaC gene, restoring the production of actinorhodin.Pristinamycin belongs to the family of streptogramin antibiotics, also called virginiamycin-like or mikamycin-like antibiotics. Streptogramins are a small and homogeneous group composed of related compounds such as pristinamycin, virginiamycin, mikamycin, and vernamycin (9, 10, 58). They are protein synthesis inhibitors (9, 10). The special feature of the family is that each member is a complex of two structurally different components exhibiting a synergistic antibacterial activity (2, 10). The two types of compounds are both macrocyclic lactone peptolides, but their structures are notably different. They belong to one of the two following distinct groups: the streptogramin A type (Sa) corresponding to polyunsaturated cyclic peptolides and the streptogramin B type (Sb) corresponding to branched cyclic hexadepsipeptides. The proportion of Sa and Sb in the complex varies from one antibiotic to another. Moreover, the major form of each component is accompanied by several structurally different minor forms.Pristinamycin, produced by Streptomyces pristinaespiralis, consists of approximately 30% pristinamycins I (PI), the Sb type molecules, and 70% pristinamycins II (PII), the Sa type molecules. In industrial strains, PII is produced mainly in two forms, PII A and PII B , in a 80:20 ratio. The difference between PII A and PII B is the presence of a dehydroproline instead of a proline in the macrocycle (Fig. 1). Thibaut et al. (57) reported high levels of conversion of radiolabelled PII B to PII A both in vivo and in vitro with several strains of Streptomyces spp. that produce pristinamycins. The same type of observation was made with Streptomyces virginiae, the producer ...

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“…erodimeric luciferase was chosen as a model enzyme because its enzymatic activity is easily detected and because of the available information about its requirements for folding and assembly after synthesis of the two subunits in E. coli cells (Waddle et al, 1987;Olsson et al, 1988;Escher et al, 1989;Flynn et al, 1993;Ziegler et al, 1993;Escher and Szalay, 1993) and rabbit reticulocyte lysate (Kruse et al, 1995), respectively. Here, we demonstrate that efficient assembly of this luciferase occurs after synthesis of the two subunits in a cell-free translation system and their concomitant transport into dog pancreas microsomes.…”

Section: ϫ3mentioning

confidence: 99%

“…The bacterial molecular chaperone GroEL was previously shown to be able to preserve the assembly-competent state of the two subunits of the heterodimeric enzyme after separate synthesis in Escherichia coli cells (Escher and Szalay, 1993;Flynn et al, 1993). Under the assumption that there is no Hsp60 homolog in the microsomal lumen, it should be interesting to see which of the microsomal chaperones can functionally substitute for Hsp60 in the microsomes.…”

Section: ϫ3mentioning

confidence: 99%

Luciferase Assembly after Transport into Mammalian Microsomes Involves Molecular Chaperones and Peptidyl-Prolyl Isomerases

Brunke

Dierks

Schlotterhose

et al. 1996

Journal of Biological Chemistry

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The assembly of a heterodimeric luciferase was studied after de novo synthesis of corresponding precursor proteins in reticulocyte lysate and concomitant transport into dog pancreas microsomes. This cytosolic luciferase from a prokaryotic organism (Vibrio harveyi) was specifically used as a model protein to investigate (i) whether the eukaryotic cytosol and the microsomal lumen have similar folding capabilities and (ii) whether the requirements of a polypeptide for certain molecular chaperones and folding catalysts are determined by the polypeptide or the intracellular compartment. The two luciferase subunits were fused to the preprolactin signal peptide. Data indicate that efficient assembly of luciferase occurs in the mammalian microsomes. Furthermore, it was observed that luciferase assembly can be separated in time from synthesis and membrane transport, depends on ATP hydrolysis, is partially sensitive to cyclosporin A and FK506, and in the absence of lumenal proteins is less efficient as compared with the presence of lumenal proteins. Thus, heterodimeric luciferase depends on functionally related molecular chaperones and folding catalysts during its assembly in either the eukaryotic cytosol or the microsomal lumen.In comparison to our knowledge about protein folding after denaturation and subsequent renaturation (Jaenicke, 1987), little is known about protein folding and assembly in the different compartments of the eukaryotic cell, following de novo synthesis of polypeptides. In general, the latter appears to be assisted by various molecular chaperones and folding catalysts (Ellis and van der Vies, 1991;Georgopoulos and Welch, 1993;Hartl et al., 1994;Kunz and Hall, 1993;Schreiber, 1991). However, the intracellular compartments are different from each other with respect to their specific set of molecular chaperones and folding catalysts (Gething and Sambrook, 1992).Recently, we studied the folding and assembly of newly synthesized proteins in the eukaryotic cytosol by employing rabbit reticulocyte lysate as a translation and folding system (Kruse et al., 1995). We asked (i) what are the kinetics of folding and assembly of two model proteins, and (ii) are ATP-dependent molecular chaperones and/or folding catalysts involved in the folding and assembly reactions? In our studies two bacterial luciferases were used as model proteins. The first luciferase was a heterodimeric enzyme (LuxAB) from Vibrio harveyi (Waddle et al., 1987;Escher et al., 1989;Flynn et al., 1993;Ziegler et al., 1993). The second luciferase was a fusion protein (Fab2) that forms a monomeric enzyme comprising LuxA and LuxB (Escher et al., 1989). Both enzymes catalyze the oxygenand FMNH 2 -dependent conversion of a long chain aldehyde to the corresponding fatty acid with concomitant emission of light (490 nm). The genes coding for LuxA, LuxB, and Fab2 were cloned into plasmids that are suitable for in vitro transcription. The plasmids were used to program coupled transcription/ translation in rabbit reticulocyte lysates. The kinetics of foldin...

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The β subunit polypeptide of Vibrio harveyi luciferase determines light emission at 42° C

Cited by 13 publications

References 27 publications

In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone

In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone

Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase

Luciferase Assembly after Transport into Mammalian Microsomes Involves Molecular Chaperones and Peptidyl-Prolyl Isomerases

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