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91.
Michael G. Hanna IV Ioanna Mela Lei Wang Robert M. Henderson Edwin R. Chapman J. Michael Edwardson Anjon Audhya 《The Journal of biological chemistry》2016,291(3):1014-1027
The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5′-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission. 相似文献
92.
Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy 下载免费PDF全文
Bacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation. DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes. Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA. Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization. This event is followed by ATP-dependent translocation and cleavage of the DNA. 相似文献
93.
Stram DO Leigh Pearce C Bretsky P Freedman M Hirschhorn JN Altshuler D Kolonel LN Henderson BE Thomas DC 《Human heredity》2003,55(4):179-190
The US National Cancer Institute has recently sponsored the formation of a Cohort Consortium (http://2002.cancer.gov/scpgenes.htm) to facilitate the pooling of data on very large numbers of people, concerning the effects of genes and environment on cancer incidence. One likely goal of these efforts will be generate a large population-based case-control series for which a number of candidate genes will be investigated using SNP haplotype as well as genotype analysis. The goal of this paper is to outline the issues involved in choosing a method of estimating haplotype-specific risk estimates for such data that is technically appropriate and yet attractive to epidemiologists who are already comfortable with odds ratios and logistic regression. Our interest is to develop and evaluate extensions of methods, based on haplotype imputation, that have been recently described (Schaid et al., Am J Hum Genet, 2002, and Zaykin et al., Hum Hered, 2002) as providing score tests of the null hypothesis of no effect of SNP haplotypes upon risk, which may be used for more complex tasks, such as providing confidence intervals, and tests of equivalence of haplotype-specific risks in two or more separate populations. In order to do so we (1) develop a cohort approach towards odds ratio analysis by expanding the E-M algorithm to provide maximum likelihood estimates of haplotype-specific odds ratios as well as genotype frequencies; (2) show how to correct the cohort approach, to give essentially unbiased estimates for population-based or nested case-control studies by incorporating the probability of selection as a case or control into the likelihood, based on a simplified model of case and control selection, and (3) finally, in an example data set (CYP17 and breast cancer, from the Multiethnic Cohort Study) we compare likelihood-based confidence interval estimates from the two methods with each other, and with the use of the single-imputation approach of Zaykin et al. applied under both null and alternative hypotheses. We conclude that so long as haplotypes are well predicted by SNP genotypes (we use the Rh2 criteria of Stram et al. [1]) the differences between the three methods are very small and in particular that the single imputation method may be expected to work extremely well. 相似文献
94.
Compartmentation of intracellular nucleotides in mammalian cells 总被引:4,自引:0,他引:4
The important role of nucleotides in cellular metabolism requires that serious consideration be given to the question of the homogeneity or inhomogeneity of nucleotide pools in cells. The purpose of this review is to summarize the existing evidence for compartmentation of nucleotide pools, discuss the limitations of this evidence, and to discuss the implications of compartmentation for the interpretation of nucleotide concentration measurements. Evidence for nucleotide compartmentation comes from the following types of evidence: compartmentation of RNA precursors; compartmentation of deoxynucleoside triphosphates; mitochondrial compartmentation; the existence of tightly bound nucleotides; pools derived from alternative synthetic routes; compartmentation in cyclic nucleotide metabolism; channeling in the synthesis of pyrimidine nucleotides; and others. The types of evidence adduced for compartmentation will be considered critically and in detail, and alternative explanations considered, as well. Implications of the data and hypotheses on nucleotide compartmentation for the interpretation of nucleotide pool measurements in various types of experiments will be discussed. 相似文献
95.
Deborah A. Occhino Elizabeth E. Wyckoff Douglas P. Henderson Thomas J. Wrona & Shelley M. Payne 《Molecular microbiology》1998,29(6):1493-1507
Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set ( tonB1, exbB1, exbD1 ) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system ( tonB2, exbB2, exbD2 ). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes ( hutBCD ), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1 . A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae , the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae . 相似文献
96.
A. C. Henderson J. L. McIlwain H. S. AlOufi A. AmbuAli 《Journal of fish biology》2006,68(6):1662-1678
Female Rhizoprionodon acutus were found to mature between 62 and 74 cm total length ( L T ) whereas males matured between 63 and 71 cm L T . The L T at 50% maturity was 64·3 cm for females and 64·7 cm for males. Litter size varied from one to six embryos, and was positively correlated with maternal L T . Female embryos outnumbered males by a ratio of 2·3:1. The size at birth was c . 37 cm L T . Full‐term embryos and post‐partum females were observed during all seasons although their occurrence was highest in spring. Spermatozoa were rarely recorded in the oviducal gland, indicating that this species does not store sperm. It was not possible to generate maturity curves for Iago omanensis but it was evident that females matured by the time they reached 35 cm and males were mature by 31 cm L T . This species displayed a clearly defined reproductive cycle with parturition occurring primarily in spring, after a gestation period of c . 1 year. Maximal embryo size was 19 cm L T while maximal litter size was 24 embryos. The oviducal gland appeared to act as a seminal receptacle and it appeared that females may utilize these stores by not mating every year. 相似文献
97.
The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor. 相似文献
98.
The goal of time-resolved crystallographic experiments is to capture dynamic "snapshots" of molecules at different stages of a reaction pathway. In recent work, we have developed approaches to determine determined light-induced conformational changes in the proton pump bacteriorhodopsin by electron crystallographic analysis of two-dimensional protein crystals. For this purpose, crystals of bacteriorhodopsin were deposited on an electron microscopic grid and were plunge-frozen in liquid ethane at a variety of times after illumination. Electron diffraction patterns were recorded either from unilluminated crystals or from crystals frozen as early as 1 ms after illumination and used to construct projection difference Fourier maps at 3.5-A resolution to define light-driven changes in protein conformation. As demonstrated here, the data are of a sufficiently high quality that structure factors obtained from a single electron diffraction pattern of a plunge-frozen bacteriorhodopsin crystal are adequate to obtain an interpretable difference Fourier map. These difference maps report on the nature and extent of light-induced conformational changes in the photocycle and have provided incisive tools for understanding the molecular mechanism of proton transport by bacteriorhodopsin. 相似文献
99.
K P McNatty K M Henderson S Lun D A Heath K Ball N L Hudson J Fannin M Gibb L E Kieboom P Smith 《Journal of reproduction and fertility》1985,73(1):109-120
A marked difference in both the function and composition of individual ovarian follicles was noted in Booroola X Romney ewes (6-7 years of age) which had previously been segregated on at least one ovulation rate record of 3-4 (F + ewes, N = 21) or less than 3 (++ ewes, N = 21). Follicles in F + ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In F+ ewes (N = 3), the presumptive preovulatory follicles were 4.4 +/- 0.5 (s.e.m.) mm in diameter and contained 2.1 +/- 0.3 X 10(6) (s.e.m.) granulosa cells, whereas in ++ ewes (N = 3), such follicles were 7.3 +/- 0.3 mm in diameter and contained 6.5 +/- 0.8 X 10(6) cells. During a prostaglandin (PG)-induced follicular phase, the secretion rate of oestradiol from ovaries containing 3 presumptive preovulatory follicles in F + ewes was similar to that from ovaries with only one such follicle in ++ ewes. We suggest that the putative 'gene effect' in F + ewes is manifested during early follicular development and that it may be mediated via an enhanced sensitivity of granulosa cells to pituitary hormones. As a consequence, the development of 3 preovulatory follicles in F + ewes may be necessary to provide a cell mass capable of producing the same quantity of oestradiol as that from one preovulatory follicle in ++ ewes. 相似文献
100.