Translocation of a lysosomal enzyme across the microsomal membrane requires signal recognition particle

Signal recognition particle (SRP), an 11S ribonucleoprotein (Walter and Blobel (1982) Nature 299, 691-698), is required for translocation of secretory proteins across microsomal membranes (Walter and Blobel (1980) Proc. Natl. Acad. Sci. USA 77, 7112-7116) and for asymmetric integration into microsomal membranes of a transmembrane protein (Anderson et al., (1982) J. Cell Biol. 93, 501-506). We demonstrate here that SRP is also required for translocation of the lysosomal protease cathepsin D across microsomal membranes.     

Model for the chemotactic response of a bacterial population.

We present a mathematical model for the motion of a bacterial population in prescribed attractant or repellent gradients. The model is suggested by the observations of Mesibov et al. (1973, J. Gen. Physiol. 62:203) and Brown and Berg (1974, Proc. Natl. Acad. Sci. U.S.A. 71:1388) who found that the sensitivity of the chemotactic response depends on the concentration of attractant. Predictions of the theory are in general agreement with the experiments of Dahlquist et al. (1972, Nat. New Biol. 236:120) and of Mesibov et al. on populations of motile bacteria in fixed attractant gradients. Additional tests of the model are proposed.     

Epstein-Barr virus DNA XII. A variable region of the Epstein-Barr virus genome is included in the P3HR-1 deletion.

The P3HR-1 subclone of Jijoye differs from Jijoye and from other Epstein-Barr virus (EBV)-infected cell lines in that the virus produced by P3HR-1 cultures lacks the ability to growth-transform normal B lymphocytes (Heston et al., Nature (London) 295:160-163, 1982; Miller et al., J. Virol. 18:1071-1080, 1976; Miller et al., Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974; Ragona et al., Virology 101:553-557, 1980). The P3HR-1 virus was known to be deleted for a region which encodes RNA in latently infected, growth-transformed cells (Bornkamm et al., J. Virol. 35:603-618, 1980; Heller et al., J. Virol. 38:632-648, 1981; King et al., J. Virol. 36:506-518, 1980; Raab-Traub et al., J. Virol. 27:388-398, 1978; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1980). This deletion is now more precisely defined. The P3HR-1 genome contains less than 170 base pairs (and possibly none) of the 3,300-base pair U2 region of EBV DNA and is also lacking IR2 (a 123-base pair repeat which is the right boundary of U2). A surprising finding is that EBV isolates vary in part of the U2 region. Two transforming EB viruses, AG876 and Jijoye, are deleted for part of the U2 region including most or all of a fragment, HinfI-c, which encodes part of one of the three more abundant cytoplasmic polyadenylated RNAs of growth-transformed cells (King et al., J. Virol. 36:506-518, 1980; King et al., J. Virol. 38:649-660, 1981; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934).     

Horizontal gene transfer in microbial genome evolution

Horizontal gene transfer is the collective name for processes that permit the exchange of DNA among organisms of different species. Only recently has it been recognized as a significant contribution to inter-organismal gene exchange. Traditionally, it was thought that microorganisms evolved clonally, passing genes from mother to daughter cells with little or no exchange of DNA among diverse species. Studies of microbial genomes, however, have shown that genomes contain genes that are closely related to a number of different prokaryotes, sometimes to phylogenetically very distantly related ones. (Doolittle et al., 1990, J. Mol. Evol. 31, 383-388; Karlin et al., 1997, J. Bacteriol. 179, 3899-3913; Karlin et al., 1998, Annu. Rev. Genet. 32, 185-225; Lawrence and Ochman, 1998, Proc. Natl. Acad. Sci. USA 95, 9413-9417; Rivera et al., 1998, Proc. Natl. Acad. Sci. USA 95, 6239-6244; Campbell, 2000, Theor. Popul. Biol. 57 71-77; Doolittle, 2000, Sci. Am. 282, 90-95; Ochman and Jones, 2000, Embo. J. 19, 6637-6643; Boucher et al. 2001, Curr. Opin., Microbiol. 4, 285-289; Wang et al., 2001, Mol. Biol. Evol. 18, 792-800). Whereas prokaryotic and eukaryotic evolution was once reconstructed from a single 16S ribosomal RNA (rRNA) gene, the analysis of complete genomes is beginning to yield a different picture of microbial evolution, one that is wrought with the lateral movement of genes across vast phylogenetic distances. (Lane et al., 1988, Methods Enzymol. 167, 138-144; Lake and Rivera, 1996, Proc. Natl. Acad. Sci. USA 91, 2880-2881; Lake et al., 1999, Science 283, 2027-2028).     

Oversized flagellar membrane protein in paralyzed mutants of Chlamydomonas reinhardrii

A mutant strain of Chlamydomonas reinhardtii is shown to possess an oversized flagellar membrane protein. The mutant has paralyzed flagella, is temperature sensitive for flagellar assembly, and has an abnormal axonemal protein composition. All phenotypes appear to derive from a single Mendelian mutation, and genetic analysis suggests that the mutation, which call ts222, is in the gene pfl. Because pf1 mutants are known to have radial-spoke defects (Piperno et al., 1977, Proc. Natl. Acad. Sci. U. S. A. 74:1600-1604; and Witman et al., 1978, J. Cell Biol. 76:729-797), a relation as yet undefined appears to exist between radial-spoke and flagellar membrane biogenesis.     

Xenopus egg lysates repair heat-generated DNA nicks with an average patch size of 36 nucleotides.

Base excision repair (BER) is an essential DNA repair pathway since it processes spontaneous (endogenous) DNA damage such as abasic sites, oxidized and alkylated bases, as well as mismatches arising from deamination of cytosine and 5-methylcytosine. Some of these lesions are repaired by the exchange of a single deoxynucleotide [Dianov, G. et al. (1992) Mol. Cell. Biol. 12, 1605-1612; Wiebauer, K. and Jiricny, J. (1990) Proc. Natl. Acad. Sci. USA, 87, 5842-5845] or a few deoxynucleotides [Matsumoto, Y. et al. (1994) Mol. Cell. Biol., 14 6187-6197]. Here we report that DNA single strand breaks induced by hyperthermic conditions are repaired with an average patch size of approximately 36 nt in Xenopus laevis egg lysates.     

Signal sequence of alkaline phosphatase of Escherichia coli.

The amino acid sequence of the signal sequence of phoA was determined by DNA sequencing by using the dideoxy chain termination technique (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, 1977). The template used was single-stranded DNA obtained from M13 on f1 phage derivatives carrying phoA, constructed by in vitro recombination. The results confirm the sequence of the first five amino acids determined by Sarthy et al. (J. Bacteriol. 139:932-939, 1979) and extend the sequence in the same reading frame into the amino terminal region of the mature alkaline phosphatase (Bradshaw et al., Proc. Natl. Acad. Sci. U.S.A., 78:3473-3477, 1981). As was predicted (Inouye and Beckwith, Proc. Natl. Acad. Sci. U.S.A. 74:1440-1444, 1977), the signal sequence was highly hydrophobic. The alteration of DNA sequence was identified for a promoter mutation that results in the expression of phoA independent of the positive control gene phoB and in insensitivity to high phosphate.     

Building a great wall around mitosis: Evolutionary conserved roles for the Greatwall/MASTL kinases in securing chromosome stability

Comment on: Voets E, et al. Cell Cycle 2010; 9:3591–3601 & Burgess A, et al. Proc Natl Acad Sci USA 2010; 107:12564–9.     

The E295K DNA polymerase beta gastric cancer-associated variant interferes with base excision repair and induces cellular transformation

Approximately 30% of human tumors examined for mutations in polymerase beta (pol beta) appear to express pol beta variant proteins (D. Starcevic, S. Dalal, and J. B. Sweasy, Cell Cycle 3:998-1001, 2004). Many of these variants result from a single amino acid substitution. We have previously shown that the K289M and I260M colon and prostate cancer variants, respectively, induce cellular transformation most likely due to sequence-specific mutator activity (S. Dalal et al., Biochemistry 44:15664-15673, 2005; T. Lang et al., Proc. Natl. Acad. Sci. USA 101:6074-6079, 2004; J. B. Sweasy et al., Proc. Natl. Acad. Sci. USA 102:14350-14355, 2005). In the work described here, we show that the E295K gastric carcinoma pol beta variant acts in a dominant-negative manner by interfering with base excision repair. This leads to an increase in sister chromatid exchanges. Expression of the E295K variant also induces cellular transformation. Our data suggest that unfilled gaps are channeled into a homology-directed repair pathway that could lead to genomic instability. The results indicate that base excision repair is critical for maintaining genome stability and could therefore be a tumor suppressor mechanism.     

Probable cloning artefacts previously interpreted as unusual leader sequences of rodent ornithine decarboxylase mRNAs--a cautionary tale

Messenger RNAs that have structurally unusual 5' leaders attract interest and provoke conjecture. Cloning and sequencing of two rodent ornithine decarboxylase (ODC) cDNAs, those for mouse [Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673-1677] and, recently as published in this journal, for rat [Van Kranen et al., Gene 60 (1987) 145-155], have indicated the presence of such features. In both cases, the leader is unusually long and contains multiple AUG start codons preceding that which encodes the N terminus of the protein. In addition, the leader of the rat clone contains a 54-nt perfect inverted repeat. Because ODC expression appears to be regulated translationally, functional implications immediately suggest themselves. Certain unusual features of the mouse cDNA have proven artefactual [Brabant et al., Proc. Natl. Acad. Sci. USA 85 (1988) 2200-2204; Katz and Kahana, J. Biol. Chem. 263 (1988) 7604-7609]. It is likely that the putative leader sequence of rat ODC cDNA also resulted from a cloning artefact.     

Milk, revealed "silent" chemistry: new mode of cycloretinal synthesis

Bovine milk is by far the most commonly consumed milk in the western world. The protein composition in milk consists of casein and whey proteins, of which β-lactoglobulin (BLG) is the principal constituent of the latter. Here we provide biochemical evidence that this milk protein, in purified form and in pasteurized store-bought milk, promotes the formation of cycloretinal (all-trans retinal dimer), and a variety of other cycloterpenals of biological relevance [Fishkin et al., Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 7091-7096; Fishkin et al., Chirality, 2004, 16, 637-641; Kim et al., Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 19273-19278]. Cycloretinal is an eye metabolite and among several toxic byproducts of the visual cycle firmly established to cause age-related macular degeneration. Experiments in rabbits further demonstrate that BLG/milk can survive the digestive system and promote this reaction in vivo [Caillard et al., Am. J. Physiol., 1994, 266(6), G1053-G1059]. Proteomic studies on age-related macular degeneration patients have detected BLG in the eye of these patients further suggesting that this milk protein could contribute to disease progression [Crabb et al., Proc. Natl. Acad. Sci. U. S. A., 2002, 99(23), 14682-14687].     

A natural oocyte component required for the reprogramming of somatic cell nuclei

Comment on: Jullien J, et al. Proc Natl Acad Sci USA 2010; 107:5483-8.     

Tubulin folding cofactor D is a microtubule destabilizing protein

A rapid switch between growth and shrinkage at microtubule ends is fundamental for many cellular processes. The main structural components of microtubules, the alphabeta-tubulin heterodimers, are generated through a complex folding process where GTP hydrolysis [Fontalba et al. (1993) J. Cell Sci. 106, 627-632] and a series of molecular chaperones are required [Sternlicht et al. (1993) Proc. Natl. Acad. Sci. USA 90, 9422-9426; Campo et al. (1994) FEBS Lett. 353, 162-166; Lewis et al. (1996) J. Cell Biol. 132, 1-4; Lewis et al. (1997) Trends Cell Biol. 7, 479-484; Tian et al. (1997) J. Cell Biol. 138, 821-823]. Although the participation of the cofactor proteins along the tubulin folding route has been well established in vitro, there is also evidence that these protein cofactors might contribute to diverse microtubule processes in vivo [Schwahn et al. (1998) Nature Genet. 19, 327-332; Hirata et al. (1998) EMBO J. 17, 658-666; Fanarraga et al. (1999) Cell Motil. Cytoskel. 43, 243-254]. Microtubule dynamics, crucial during mitosis, cellular motility and intracellular transport processes, are known to be regulated by at least four known microtubule-destabilizing proteins. OP18/Stathmin and XKCM1 are microtubule catastrophe-inducing factors operating through different mechanisms [Waters and Salmon (1996) Curr. Biol. 6, 361-363; McNally (1999) Curr. Biol. 9, R274-R276]. Here we show that the tubulin folding cofactor D, although it does not co-polymerize with microtubules either in vivo or in vitro, modulates microtubule dynamics by sequestering beta-tubulin from GTP-bound alphabeta-heterodimers.     

Actin in the ciliated protozoan Climacostomum virens: purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis.

The anti-actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335-2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti-alpha-smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787-2796, 1986) nor with an anti-alpha-sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515-531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity-purified actin also reacts with mab JLA20. Two-dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KCl, the affinity-purified actin polymerizes into filaments as shown by electron microscopy after negative staining.     

The interaction of DNA polymerase III and the product of the Escherichia coli mutator gene, mutD

A comparison of DNA polymerase III core enzyme (McHenry, C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748-1753) prepared from wild type Escherichia coli and a strain harboring the mutator gene, mutD5 (Degnen, G. E., and Cox, E. C. (1974) J. Bacteriol. 17, 477-487) has revealed several differences in their properties. Among these are alterations in the heat stability, divalent cation requirement, pH optimum, 3'----5'-single strand exonuclease activity, and DNA-dependent conversion of a deoxynucleoside triphosphate to its corresponding monophosphate ("turnover"). The decrease in the 3'-single strand exonuclease and turnover indicate a defect in the editing function of the mutD strain, which is at least in part responsible for the high spontaneous mutation rate in mutD. Transformation of mutD by a hybrid plasmid, pRD3, constructed from an EcoRI restriction fragment of E. coli and pBR322, cures mutD of its abnormally high mutation rate, and simultaneously restores its 3'-exonuclease activity. These observations are consistent with the notion that the mutD gene product is a subunit of DNA polymerase III, and it either contains the catalytic site for the 3'-exonuclease or modulates its activity. From a consideration of the known molecular weights of the subunits in DNA polymerase III core (McHenry C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748-1753) the molecular weights of the two proteins translated in maxicells transformed with pRD3, and from a comparison of our results with those obtained with the mutator dnaQ (Horiuchi, T., Maki, H., Maruyama, M., and Sekiguchi, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3770-3774) and the work of Cox and Horner (Cox, E. C., and Horner, D. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2295-2299) as well as Echols et al. (Echols, H., Lu, C., and Burgers, P. M. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2189-2192) we tentatively assign the mutD gene product to the epsilon subunit of DNA polymerase III.     

Reading the three-dimensional structure of lattice model-designed proteins from their amino acid sequence.

While all the information required for the folding of a protein is contained in its amino acid sequence, one has not yet learned how to extract this information to predict the detailed, biological active, three-dimensional structure of a protein whose sequence is known. Using insight obtained from lattice model simulations of the folding of small proteins (fewer than 100 residues), in particular of the fact that this phenomenon is essentially controlled by conserved contacts (Mirny et al., Proc Natl Acad Sci USA 1995;92:1282) among (few) strongly interacting ("hot") amino acids (Tiana et al., J Chem Phys 1998;108:757-761), which also stabilize local elementary structures formed early in the folding process and leading to the (postcritical) folding core when they assemble together (Broglia et al., Proc Natl Acad Sci USA 1998;95:12930, Broglia & Tiana, J Chem Phys 2001;114:7267), we have worked out a successful strategy for reading the three-dimensional structure of lattice model-designed proteins from the knowledge of only their amino acid sequence and of the contact energies among the amino acids.     

Absorption and circular dichroism spectra of different forms of mushroom tyrosinase.

The circular dichroism spectrum of resting mushroom tyrosinase between 800 and 400 nm showed two bands at 755, and 653 nm. The CD spectrum of resting tyrosinase between 400 and 250 nm showed oxygen-sensitive changes at 350 nm upon treatment of tyrosinase with hydroxylamine or hydrogen peroxide. These were similar to changes observed on regeneration of aged hemocyanin by similar procedures. A structural relationship between the active sites of hydroxylamine- or hydrogen peroxide-treated tyrosinase and hemocyanin is suggested by these observations, confirming inferences based upon other studies (Jolly, Jr., R.L., Evans, L.H., Makino, N. and Mason, H.S. (1974) J. Biol. Chem. 249, 335-345 and Schoot Uiterkamp, A.J.M. and Mason, H.S. (1973) Proc. Natl. Acad, Sci. U.S. 70, 993-996).     

Embryonal cell surface recognition. Extraction of an active plasma membrane component.

Plasma membranes obtained from different neural regions of the chicken embryo have previously been shown to specifically bind to homotypic cells and prevent cell aggregation (Merrell, R., and Glaser, L. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 2794-2798). Proteins responsible for the specific inhibition of cell aggregation have been solubilized from the plasma membrane of neural retina and optic tectum by delipidation with acetone followed by extraction with lithium diiodosalicylate. The extracts show the same regional and temporal specificity as previously shown for plasma membrane recognition by the same cells (Gottlieb, D. I., Merrell, R., and Glaser, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1800-1802). Two micrograms of the most purified protein fraction inhibits the aggregation of 2.5 times 10(-4) cells under standard assay conditions. This represents a 20-fold increase in specific activity compared to whole membranes.     

Bending and curvature calculations in B-DNA.

A simple program, BEND, has been written to calculate the magnitude of local bending and macroscopic curvature at each point along an arbitrary B-DNA sequence, using any desired bending model that specifies values of twist, roll and tilt as a function of sequence. The program has been used to evaluate six different DNA bending models in three categories. Two are bent non-A-tract models: (a) A new model based on the nucleosome positioning data of Satchwell et al 1986 (J. Mol. Biol. 191, 659-675), (b) The model of Calladine et al 1988 (J. Mol. Biol. 201, 127-137). Three are bent A-tract models: (c) The wedge model of Bolshoy et al 1991 (Proc. Natl. Acad. Sci. USA 88, 2312-2316), (d) The model of Cacchione et al 1989 (Biochem. 28, 8706-8713), (e) A reversed version of model (b). The last is a junction model: (f) The model of Koo & Crothers 1988 (Proc. Natl. Acad. Sci. USA 85, 1763-1767). Although they have widely different assumptions and values for twist, roll and tilt, all six models correctly predict experimental A-tract curvature as measured by gel retardation and cyclization kinetics, but only the new nucleosome positioning model is successful in predicting curvature in regions containing phased GGGCCC sequences. This model--showing local bending at mixed sequence DNA, strong bends at the sequence GGC, and straight, rigid A-tracts--is the only model consistent with both solution data from gel retardation and cyclization kinetics and structural data from x-ray crystallography.     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.