The activity of DnaK (Hsp70) chaperones in assisting protein folding relies on DnaK binding and ATP-controlled release of protein substrates. The ATPase activity of DnaK is tightly controlled by the nucleotide exchange factor GrpE. We find that GrpE interacts stably with the amino-terminal ATPase domain of DnaK. Analysis of the mutant DnaK756 protein, which has a lower affinity for GrpE, reveals a role for residue Gly 32 in GrpE binding. Gly 32 is located in an exposed loop near the nucleotide binding site of DnaK. Deletion of this loop prevents stable GrpE binding, ATPase stimulation by GrpE, and DnaK chaperone activity. Conservation of this loop within the Hsp70 family suggests that cooperation between Hsp70 and GrpE-like proteins may be a general feature of this class of chaperone.
The first discovery of an Hsp70 chaperone gene was the isolation of an Escherichia coli mutant, dnaK756, which rendered the cells resistant to lytic infection with bacteriophage . The DnaK756 mutant protein has since been used to establish many of the cellular roles and biochemical properties of DnaK. DnaK756 has three glycine-to-aspartate substitutions at residues 32, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the substrate-binding domain, interdomain communication, and, consequently, defects in chaperone activity. To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the amino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 468) substitutions. The amino-terminal substitution interfered with the GrpE-stimulated ATPase activity. The carboxyl-terminal mutations (i) affected stability and function of the substrate-binding domain, (ii) caused a 10-fold elevated ATP hydrolysis rate, but (iii) did not severely affect domain coupling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutions both in vivo and in vitro. In the in vitro refolding of denatured firefly luciferase, the defect of the DnaK variant carrying the amino-terminal substitution results from its inability to release, upon GrpE-mediated nucleotide exchange, bound luciferase in a folding competent state. Our results indicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal substrate binding, whereas the tight kinetic control of substrate dissociation by GrpE is essential.In 1977, Georgopoulos (1) reported a mutation, groPC756 later termed dnaK756, which conferred resistance to Escherichia coli against lytic infection by bacteriophage (2, 3). This was the first discovery of a member of the ubiquitous Hsp70 chaperone family (4 -6), the DnaK heat shock protein, and the DnaK756 mutant protein has since been widely used to dissect cellular functions and biochemical features of DnaK. DnaK is now known to play central roles in assisting folding processes in the entire life span of proteins in the E. coli cytosol (7). In the present study we dissected the functional defects of the classical DnaK756 mutant protein, which provided insights into mechanistic features of DnaK.DnaK consists of an amino-terminal, highly conserved ATPase domain (residues 1-385), an adjacent, conserved substrate-binding domain (residues 393-537), and a more diverse carboxyl-terminal domain (residues 538 -638) of unclear function. Substrate interactions of the substrate-binding domain are controlled by the nucleotide state of the ATPase domain, thus indicating functional coupling of the two domains (8). The ATP state is characterized by low affinity and fast exchange rates for substrates, whereas the ADP state possesses high affinity and low exchange rates for substrates (9, 10). ATP hydrolysis is a key s...
The DNA content per cell of two tested auxotrophic mutants of Pichia guilliermondii was diminished in comparison to their wild-type strains. After protoplast fusion between these mutitnta the majority of hybrids did not show the expected amount of DNA. In some hybrids it was only slightly higher than in the wild-type strains, although heterozygosity for markers of three chromosomes could be established.The protein content per cell of most strains was positively correlated wit.h the DNA content, but generally more affected as the DNA content by the induction of mutations and protoplast fusion. The protein/DNA ratio of the two mutants was strongly reduced, while this ratio was somewhat enhanced in one of the hybrids in comparison to the wild-type strains and the majority of hybrids. Apparently, thc reduced protein content per cell in the mutants seemed to be partially compensated in the hybrids. FXRENCZY 1977, BOTTCHER et al. 197.8, MARAZ et al. 1978, VALLIN u. FERENCZY 1978. Zur Hybridisierung von Mikroorganismen wurde in den letztenDariiber hinaus sind biochemische Konsequenzen der Protoplastenfusion bisher wenig untersucht worden (ANNE u. PEBERDY 1981).Wir haben bei der Futterhefe Pichia guilliermondii die Korrelation zwischen DNAund Proteingehalt pro Zelle bei Fusionshybriden und ihren Ausgangsstammen untersucht. Wciterhin wixrde in diesen Stammen das Muster ausgewahltcr Enzyme bestimmt. I n der vorliegenden Arbeit sind zunachst die Ergehnisse der genetischen Charakterisierung sowie der DNA-und Proteinbestimmungen beschrieben. Material und Methoden
Purpose Comparison of puncture deviation and puncture duration between computed tomography (CT)- and C-arm CT (CACT)-guided puncture performed by residents in training (RiT). Methods In a cohort of 25 RiTs enrolled in a research training program either CT- or CACT-guided puncture was performed on a phantom. Prior to the experiments, the RiT’s level of training, experience playing a musical instrument, video games, and ball sports, and self-assessed manual skills and spatial skills were recorded. Each RiT performed two punctures. The first puncture was performed with a transaxial or single angulated needle path and the second with a single or double angulated needle path. Puncture deviation and puncture duration were compared between the procedures and were correlated with the self-assessments. Results RiTs in both the CT guidance and CACT guidance groups did not differ with respect to radiologic experience (p = 1), angiographic experience (p = 0.415), and number of ultrasound-guided puncture procedures (p = 0.483), CT-guided puncture procedures (p = 0.934), and CACT-guided puncture procedures (p = 0.466). The puncture duration was significantly longer with CT guidance (without navigation tool) than with CACT guidance with navigation software (p < 0.001). There was no significant difference in the puncture duration between the first and second puncture using CT guidance (p = 0.719). However, in the case of CACT, the second puncture was significantly faster (p = 0.006). Puncture deviations were not different between CT-guided and CACT-guided puncture (p = 0.337) and between the first and second puncture of CT-guided and CACT-guided puncture (CT: p = 0.130; CACT: p = 0.391). The self-assessment of manual skills did not correlate with puncture deviation (p = 0.059) and puncture duration (p = 0.158). The self-assessed spatial skills correlated positively with puncture deviation (p = 0.011) but not with puncture duration (p = 0.541). Conclusion The RiTs achieved a puncture deviation that was clinically adequate with respect to their level of training and did not differ between CT-guided and CACT-guided puncture. The puncture duration was shorter when using CACT. CACT guidance with navigation software support has a potentially steeper learning curve. Spatial skills might accelerate the learning of image-guided puncture. Key Points: Citation Format
We investigated the enzyme patterns of two wild-type strains, two auxotrophic mutants and some fusion hybrids of Pichia guilliermondii after the electrophoretic separation of their crude cell extracts in dependence on the cultivation on mineral salt medium with different carbon sources. The wild-type strains showed a constitutive synthesis of one NAD-specific and some NADP-specific enzymes and their ability to oxidize primary alcohols with a chain length from C, to C,. After the induction of mutants auxotroph for ,.mino acids, nicotinic acid or adenine we found different enzyme patterns, too. Prototrophic fusion hybrids obtained via protoplast fusion of the two double auxotrophic mutants showed behaviour either characteristic for the wild-type strains or characteristics of their parental strains and in some cases also combination of parental enzyme patterns.The behaviour of the hybrids seems to be dependent on the quantity of DNA transferred into each fusion hybrid from the parental mutant strains.
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