Verprolin cytokinesis function mediated by the Hof one trap domain

In budding yeast, partitioning of the cytoplasm during cytokinesis can proceed via a pathway dependent on the contractile actomyosin ring, as in other eukaryotes, or alternatively via a septum deposition pathway dependent on an SH3 domain protein, Hof1/Cyk2 (the yeast PSTPIP1 ortholog). In dividing yeast cells, Hof1 forms a ring at the bud neck distinct from the actomyosin ring, and this zone is active in septum deposition. We previously showed the yeast Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) ortholog, verprolin/Vrp1/End5, interacts with Hof1 and facilitates Hof1 recruitment to the bud neck. A Vrp1 fragment unable to interact with yeast WASP (Las17/Bee1), localize to the actin cytoskeleton or function in polarization of the cortical actin cytoskeleton nevertheless retains function in Hof1 recruitment and cytokinesis. Here, we show the ability of this Vrp1 fragment to bind the Hof1 SH3 domain via its Hof one trap (HOT) domain is critical for cytokinesis. The Vrp1 HOT domain consists of three tandem proline-rich motifs flanked by serines. Unexpectedly, the Hof1 SH3 domain itself is not required for cytokinesis and indeed appears to negatively regulate cytokinesis. The Vrp1 HOT domain promotes cytokinesis by binding to the Hof1 SH3 domain and counteracting its inhibitory effect.     

The suppression of one plant virus by another

Severe etch virus prevents die multiplication of potato virus Y and Hyoscyamus virus 3 and replaces them even in plants in which they are established. Mild etch virus reduces the concentration of potato virus Y but does not suppress it completely. Cucumber virus 1 multiplies normally in mixed infections with any of the three other insect-transmitted viruses. Possible implications of these results on the mechanism of virus multiplication are discussed; it is suggested that these viruses inactivate in cell sap at approximately the same rate as they denature in vitro.
No differences were found between the stability of antibodies to viruses with different properties.     

Plasmid fusions mediated by one end of TnA

We have observed plasmid fusions in a recA background mediated by a single end of TnA. These occur when transposase is provided either in cis or in trans. Insertions of the plasmid carrying the TnA inverted repeat sequence occur at many sites in the target plasmid. The point of fusion on the plasmid carrying TnA sequences always appears to be located in the region which carries the TnA inverted repeat sequence. In contrast to the transposition of an intact TnA element, plasmid fusions mediated by one end of TnA are very rare events. The implications of our results for models of transposition are discussed.     

The two membrane segments of leader peptidase partition one by one into the lipid bilayer via a Sec/YidC interface

We have addressed the mechanism of insertion of both transmembrane segments (TMs) of leader peptidase, a double-spanning protein, into the Escherichia coli inner membrane. Using photo-crosslinking, the first TM (H1) was shown to insert at a Sec-translocon/YidC interface in a fixed orientation. H1 lost its contacts with the Sec-translocon and gained access to lipids near YidC soon after complete exposure outside the ribosome. Following lipid integration, it moved away from the Sec/YidC insertion site. The second TM (H2) inserted and interacted with SecY and YidC in a similar transient fashion. The data are consistent with a linear integration model in which the TMs of polytopic inner membrane proteins move one by one from a Sec/YidC insertion site into the lipid bilayer. We propose that YidC assists the lipid partitioning of single TMs.     

Dextran augments delayed-type hypersensitivity by interrupting one limb of the suppressor cascade

We have studied the immunomodulatory effect of dextran on the development of delayed-type contact hypersensitivity to a hapten in mice. Administration of an optimal dose of dextran 2 hours before applying picryl chloride to abdominal skin caused a twofold rise in the level of hapten-specific DTH. A study of the kinetics of development of DTH under the influence of dextran showed that comparable levels of response could be seen 2 days earlier in treated than in untreated mice, i.e., on the third day in contrast to the fifth day after sensitization. The peak of the responses, while greater in dextran-treated mice than in normal controls, remained the same at 5 days. Adoptive transfer studies revealed that comparable levels of DTH were conferred upon recipient mice by half the number of splenic cells from dextran-treated mice than that required from normal sensitized mice. Because several suppressor mechanisms are known to down-regulate DTH, we have studied dextran's effect on the neutralization of these systems as a possible explanation for its enhancing capabilities. Detailed examination was made of dextran's effect on the two suppressor T cells, Ts1 and Ts3, that act in tandem as well as its effect on the Ts1 and macrophage that work in combination. Both systems depress the efferent limb of DTH. We have found that dextran blocks the Ts1-macrophage pathway that controls DTH. Ts1 was found to arise normally in mice pretreated with dextran. Furthermore, Ts1 from dextran-treated mice produced TsF1 normally. However, we have found that dextran interferes with the production of macrophage suppressor factor (M phi-SF). Interference was partial when dextran was introduced during the interval in which macrophages were being armed with TsF1, and it was complete when dextran was put with pre-armed macrophages before they were triggered with antigen for production of M phi-SF. On the other hand, the Ts1-Ts3 limb of suppression remained unaffected by exposure to the immunomodulator. We found Ts3 arose normally in hapten-sensitized mice that had been pretreated with dextran. In addition, Ts3 became armed with TsF1 in vitro in the presence of dextran since the cells functioned properly to suppress mature DTH effector cells. Finally, TsF3 was able to act in vitro upon DTH effector cells despite the presence of dextran.(ABSTRACT TRUNCATED AT 400 WORDS)     

Torque generation by one of the motor subunits of heterotrimeric kinesin-2

Heterotrimeric kinesin-2 motors [1] and [2] transport intraflagellar transport (IFT)-particles from the base to the tip of the axoneme to assemble and maintain cilia [3], [4], [5], [6], [7], [8], [9] and [10]. These motors are distinct in containing two non-identical motor subunits together with an accessory subunit [1], [11], [12], [13], [14] and [15]. We evaluated the significance of this organization by comparing purified wild type kinesin-2 holoenzymes that support IFT in vivo, with mutant trimers containing only one type of motor domain that do not support IFT in vivo. In motility assays, wild type kinesin-2 moved microtubules (MTs) at a rate intermediate between the rates supported by the two mutants. Interestingly, one of the mutants, but not the other mutant or the wild type protein, was observed to drive a persistent counter-clock-wise rotation of the gliding MTs. Thus one of the two motor domains of heterotrimeric kinesin-2 exerts torque as well as axial force as it moves along a MT, which may allow kinesin-2 to control its circumferential position around a MT doublet within the cilium.     

Tetracycline resistance determined by pBR322 is mediated by one polypeptide

Only one polypeptide specified by plasmid pBR322 is necessary to determine tetracycline resistance. Small deletions in pBR322 constructed in vitro which result in the lack of ability to confer tetracycline resistance in vivo also result in the absence or alteration of this polypeptide in vivo. Other deletions define the extent of material necessary to encode this polypeptide. A correction to the DNA sequence of the tetracycline resistance cistron has been determined which confirms these observations.     

Artemia habitats: Ion concentrations tolerated by one superspecies

The geographic distribution, history, and ionic composition of habitats of Artemia franciscana are reviewed with emphasis on habitats with extreme values for ionic concentrations or ionic ratios: a) high-chloride waters (sea water salterns and Zuni and Great Salt Lakes); b) high-sulfate lakes in Saskatchewan (Chaplin and Little Manitou) and on the Okanogan plateau of Washington (Penley Lake complex); and c) high-carbonate habitats in Nevada (Fallon), in California (Mono Lake) and in the Nebraska sandhills (Jesse and Antioch).First-instar nauplii from populations representative of each of these three habitat clusters were tested for tolerance of potassium (0–5 g K l^-1), magnesium (0–1.3 g Mg l^-1), and calcium (0\2–0.6 g Ca l^-1). Viabilities were recorded until survivors reached adulthood in pairs of simple defined synthetic culture media which differed in only one parameter. Eight populations showed four levels of tolerance of high potassium. Of four populations tested, all had high viability and fertility in media lacking potassium (above the level in the yeast diet). Artemia from sea water salterns or from Zuni, Chaplin, or Great Salt Lakes could not tolerate low levels of calcium (<20 mg l^-1). This accounts for their inability to tolerate hypersaline high-carbonate waters. Mono and Fallon nauplii had high viability and fertility in media with low levels of calcium (0–10 mg l^-1) but lacking magnesium. They could not survive for seven days, however, in low-calcium (< 10 mg l^-1) media that contained moderate amounts of magnesium (1.3 g l^-1), indicating that magnesium interferes with utilization of low levels of calcium.For each of the three cations, the range of concentrations encountered by each population in the habitat is narrower than the range affording high viability in laboratory media. As expected, the midpoints of the two ranges are sometimes similar. In many cases, however, the narrower range of ionic concentrations reported for lake water is at the end of the range affording high viability in the laboratory.     

Negative regulation of cell proliferation by mevalonate or one of the mevalonate phosphates

The role of mevalonate and its products in the regulation of cellular proliferation was examined using 6-fluoromevalonate (Fmev), a compound that blocks the conversion of mevalonate pyrophosphate to isopentenyl pyrophosphate. Fmev suppressed DNA synthesis by a variety of transformed and malignant T cell, B cell, and myeloid cell lines. In contrast to results previously reported with mitogen-stimulated human peripheral blood T cell DNA synthesis, low concentrations of low density lipoprotein (LDL) alone could not restore proliferation to these cell lines. The same concentrations of LDL were able to provide sufficient cholesterol and support the growth of all cell lines when mevalonate synthesis was blocked with a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, lovastatin. Fmev-mediated inhibition was totally prevented in some but not all cell lines when the concentration of exogenous LDL was increased 5-10-fold above that required to permit proliferation of lovastatin-blocked cells. Residual HMG-CoA reductase activity of cells cultured with LDL inversely correlated with the restoration of growth to Fmev-blocked cultures. Confirmation of the critical role of HMG-CoA reductase activity and mevalonate synthesis in the inhibition of cellular proliferation by Fmev was obtained by demonstrating that the specific inhibitor of this enzyme, lovastatin, restored proliferation of Fmev-blocked cells. Furthermore, supplementation of cultures with mevalonate, the product of HMG-CoA reductase activity, markedly inhibited proliferation of Fmev-blocked cells. These findings indicate that mevalonate or one of the mevalonate phosphates, which accumulates in Fmev-blocked cells, is a critical negative regulator of cellular proliferation.     

Endonuclease VII has two DNA-binding sites each composed from one N- and one C-terminus provided by different subunits of the protein dimer.

Endonuclease VII (endo VII) is a Holliday structure-resolving enzyme of bacteriophage T4. Its activity depends on dimerization, DNA binding and hydrolysis of two phosphodiester bonds flanking the Holliday junction. We analysed the DNA-binding activity of truncated monomeric and covalently linked dimeric endo VII proteins. We show that both ends of endo VII are involved in DNA binding. In particular, the C-terminus of one subunit interacts with the N-terminus of the other subunit, constituting one DNA-binding site; the other two termini form the second binding site of the dimer. One binding site is sufficient to bind cruciform DNA. The concerted mechanism involving termini from different subunits ensures that only dimers bind to Holliday structures, thus providing two catalytic centres which introduce two cleavages in opposite strands. This is a precondition for precise resolution of Holliday structures.