Role of a Conserved Glutamate Residue in the \u3cem\u3eEscherichia coli\u3c/em\u3e SecA ATPase Mechanism

Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the “DEVD” Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018–2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s–1versus 27 s–1, respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439–17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis

Saccharomyces cerevisiae Rad9 Acts as a Mec1 Adaptor to Allow Rad53 Activation

SummaryBackground: The DNA damage checkpoint is a protein kinase-based signaling system that detects and signals physical alterations in DNA. Despite having identified many components of this signaling cascade, the exact mechanisms by which checkpoint kinases are activated after DNA damage, as well as the role of the checkpoint mediators, remain poorly understood.Results: To elucidate the mechanisms that underlie the MEC1 and RAD9-dependent activation of Rad53, the Saccharomyces cerevisiae ortholog of Chk2, we mapped and characterized in vivo phosphorylation sites present on Rad53 after DNA damage by mass spectrometry. We find that Rad53 requires for its activation multisite phosphorylation on a number of typical and atypical Mec1 phosphorylation sites, thus confirming that Rad53 is a direct target of Mec1, the mammalian ATR homolog. Moreover, by using biochemical reconstitution experiments, we demonstrate that efficient and direct phosphorylation of Rad53 by Mec1 is only observed in the presence of purified Rad9, the archetypal checkpoint mediator. We find that the stimulatory activity of Rad9 requires a phospho- and FHA-dependent interaction with Rad53, which allows Rad53 to be recognized as a substrate for Mec1.Conclusions: Our results indicate that Rad9 acts as a bona fide signaling adaptor that enables Rad53 phosphorylation by Mec1. Given the high degree of conservation of checkpoint signaling in eukaryotes, we propose that one of the critical functions of checkpoint mediators such as MDC1, 53BP1, or Brca1 is to act as PIKK adaptors during the DNA damage response

The characterization of amphibian nucleoplasmins yields new insight into their role in sperm chromatin remodeling

BACKGROUND: Nucleoplasmin is a nuclear chaperone protein that has been shown to participate in the remodeling of sperm chromatin immediately after fertilization by displacing highly specialized sperm nuclear basic proteins (SNBPs), such as protamine (P type) and protamine-like (PL type) proteins, from the sperm chromatin and by the transfer of histone H2A-H2B. The presence of SNBPs of the histone type (H type) in some organisms (very similar to the histones found in somatic tissues) raises uncertainty about the need for a nucleoplasmin-mediated removal process in such cases and poses a very interesting question regarding the appearance and further differentiation of the sperm chromatin remodeling function of nucleoplasmin and the implicit relationship with SNBP diversity The amphibians represent an unique opportunity to address this issue as they contain genera with SNBPs representative of each of the three main types: Rana (H type); Xenopus (PL type) and Bufo (P type). RESULTS: In this work, the presence of nucleoplasmin in oocyte extracts from these three organisms has been assessed using Western Blotting. We have used mass spectrometry and cloning techniques to characterize the full-length cDNA sequences of Rana catesbeiana and Bufo marinus nucleoplasmin. Northern dot blot analysis shows that nucleoplasmin is mainly transcribed in the egg of the former species. Phylogenetic analysis of nucleoplasmin family members from various metazoans suggests that amphibian nucleoplasmins group closely with mammalian NPM2 proteins. CONCLUSION: We have shown that these organisms, in striking contrast to their SNBPs, all contain nucleoplasmins with very similar primary structures. This result has important implications as it suggests that nucleoplasmin's role in chromatin assembly during early zygote development could have been complemented by the acquisition of a new function of non-specifically removing SNBPs in sperm chromatin remodeling. This acquired function would have been strongly determined by the constraints imposed by the appearance and differentiation of SNBPs in the sperm

Influence of duration of dietary vitamin E supplementation on swine growth performance and carcass quality

Supplementing medium-lean genotype pigs with supranutritional concentrations of dietary vitamin E (91 IU d-a-tocopheryl acetate/lb of feed) for as long as 70 d during the finishing phase was not effective in improving swine performance, feeding characteristics, and 24 h loin muscle quality. However, lower carcass temperatures obtained by spray chilling pork sides at O°C versus 4.4°C had a beneficial effect on 24 h carcass quality by improving marbling and lean firmness scores and reducing loin muscle moisture exudate. Overall, 24 h pork carcass quality was impacted more by chill rate than dietary vitamin E supplementation.; Swine Day, Manhattan, KS, November 19, 199

RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration

Increasing peptide identifications and decreasing search times for ETD spectra by pre-processing and calculation of parent precursor charge

<p>Abstract</p> <p>Background</p> <p>Electron Transfer Dissociation [ETD] can dissociate multiply charged precursor polypeptides, providing extensive peptide backbone cleavage. ETD spectra contain charge reduced precursor peaks, usually of high intensity, and whose pattern is dependent on its parent precursor charge. These charge reduced precursor peaks and associated neutral loss peaks should be removed before these spectra are searched for peptide identifications. ETD spectra can also contain ion-types other than c and z<b>˙</b>. Modifying search strategies to accommodate these ion-types may aid in increased peptide identifications. Additionally, if the precursor mass is measured using a lower resolution instrument such as a linear ion trap, the charge of the precursor is often not known, reducing sensitivity and increasing search times. We implemented algorithms to remove these precursor peaks, accommodate new ion-types in noise filtering routine in OMSSA and to estimate any unknown precursor charge, using Linear Discriminant Analysis [LDA].</p> <p>Results</p> <p>Spectral pre-processing to remove precursor peaks and their associated neutral losses prior to protein sequence library searches resulted in a 9.8% increase in peptide identifications at a 1% False Discovery Rate [FDR] compared to previous OMSSA filter. Modifications to the OMSSA noise filter to accommodate various ion-types resulted in a further 4.2% increase in peptide identifications at 1% FDR. Moreover, ETD spectra when searched with charge states obtained from the precursor charge determination algorithm is shown to be up to 3.5 times faster than the general range search method, with a minor 3.8% increase in sensitivity.</p> <p>Conclusion</p> <p>Overall, there is an 18.8% increase in peptide identifications at 1% FDR by incorporating the new precursor filter, noise filter and by using the charge determination algorithm, when compared to previous versions of OMSSA.</p

Par3 Controls Epithelial Spindle Orientation by aPKC-Mediated Phosphorylation of Apical Pins

SummaryBackgroundFormation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, misorientation creates multiple lumens.ResultsWe now show that silencing of the polarity protein Par3 causes spindle misorientation in Madin-Darby canine kidney cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3 or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects.ConclusionsThe Par3 and aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation