Ssp1 promotes actin depolymerization and is involved in stress response and new end take-off control in fission yeast

The ssp1 gene encodes a protein kinase involved in alteration of cell polarity in Schizosaccharomyces pombe. ssp1 deletion causes stress sensitivity, reminiscent of defects in the stress-activated MAP kinase, Spc1; however, the two protein kinases do not act through the same pathway. Ssp1 is localized mainly in the cytoplasm, but after a rise in external osmolarity it is rapidly recruited to the plasma membrane, preferentially to active growth zones and septa. Loss of Ssp1 function inhibits actin relocalization during osmotic stress, in cdc3 and cdc8 mutant backgrounds, and in the presence of latrunculin A, implicating Ssp1 in promotion of actin depolymerization. We propose a model in which Ssp1 can be activated independently of Spc1 and can partially compensate for its loss. The ssp1 deletion mutant exhibited monopolar actin distribution, but new end take-off (NETO) could be induced in these cells by exposure to KCl or to latrunculin A pulse treatment. This treatment induced NETO in cdc10 cells arrested in G1 but not in tea1 cells. This suggests that cells that contain intact cell end markers are competent to undergo NETO throughout interphase, and Ssp1 is involved in generating the NETO stimulus by enlarging the actin monomer pool.     

A rapid method for exploring the protein structure universe

We have developed an automatic protein fingerprinting method for the evaluation of protein structural similarities based on secondary structure element compositions, spatial arrangements, lengths, and topologies. This method can rapidly identify proteins sharing structural homologies as we demonstrate with five test cases: the globins, the mammalian trypsinlike serine proteases, the immunoglobulins, the cupredoxins, and the actinlike ATPase domain-containing proteins. Principal components analysis of the similarity distance matrix calculated from an all-by-all comparison of 1,031 unique chains in the Protein Data Bank has produced a distribution of structures within a high-dimensional structural space. Fifty percent of the variance observed for this distribution is bounded by six axes, two of which encode structural variability within two large families, the immunoglobulins and the trypsinlike serine proteases. Many aspects of the spatial distribution remain stable upon reduction of the database to 140 proteins with minimal family overlap. The axes correlated with specific structural families are no longer observed. A clear hierarchy of organization is seen in the arrangement of protein structures in the universe. At the highest level, protein structures populate regions corresponding to the all-alpha, all-beta, and alpha/beta superfamilies. Large protein families are arranged along family-specific axes, forming local densely populated regions within the space. The lowest level of organization is intrafamilial; homologous structures are ordered by variations in peripheral secondary structure elements or by conformational shifts in the tertiary structure.     

A new sulfated beta-galactan from clams with anti-HIV activity

A new polysaccharide composed of galactan sulfate with a beta-(1-->3)-glycosidic linkage has been isolated from the marine clam species Meretrix petechialis. The polysaccharide was homogeneous in its composition containing D-galactose. The glycosidic linkage was examined by 2D DQF-COSY and 2D NOESY spectroscopy. The coupling constant of anomeric proton was 7.8 Hz, suggesting a beta-galacto configuration. The downfield shift of H-2 of galactose residue demonstrated the presence of 2-O-sulfonate group. TQF-COSY confirmed that the C-6 position was substituted with a sulfonate group. The anti-HIV activity of the polysaccharides has been evaluated by the inhibition of syncytia formation. The fusion index and percentage fusion inhibition of sulfated galactan were 0.34 and 56% at 200 micrograms/mL.     

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.     

Characterization of the Fourth α Isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K _D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na⁺ and K⁺. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999     

Antibody analysis of the localisation, expression and stability of HlyD, the MFP component of the E. coli haemolysin translocator

HlyD has a single transmembrane domain (residues 59-80) and a large periplasmic domain, and is essential for the secretion of haemolysin from Escherichia coli. Using an antibody raised against HlyD, the protein was localised to the cell envelope by immunofluorescence and to the cytoplasmic membrane by sucrose gradient analysis. We have examined the stability of this protein in the presence and absence of other putative components of the translocator, HlyB and TolC. HlyD is normally highly stable but in the absence of TolC, the steady-state level of HlyD is greatly reduced and the protein has a half-life at 37° C of 36 min. In the absence of HlyB, HlyD is also unstable and specific degradation products are detected, which co-fractionate with the inner membrane, indicating in this case limited cleavage at specific sites. However, the effect of removing both HlyB and TolC is not additive. On the contrary, in the absence of both HlyB and TolC the half-life of HlyD is approximately 110 min. This result shows that in the presence of HlyB removal of TolC renders HlyD more unstable than it is in the absence of both HlyB and TolC. This suggests that the presence of HlyB induces a structural change in HlyD. In addition, HlyB itself appears to be less stable in the absence of HlyD. These results are consistent with an interaction between HlyD/TolC and HlyB/HlyD. A derivative of HlyD, HlyD22, lacking the 40 N-terminal residues of HlyD assembles into the inner membrane displaying the same stability with and without HlyB as wild type HlyD does. This N-terminal region therefore appears to play no role in stable localisation but is involved in secretion, since HlyD22 is completely secretion defective. Modification of the C-terminus on the other hand completely destabilised the molecule and HlyD was not detectable in the envelope. Secretion of active haemolysin is limited to a brief period during mid to late exponential phase. In contrast, HlyD is apparently synthesised constitutively throughout the growth phase, demonstrating that the production of this component of the translocator is not the limiting factor for growth phase-dependent secretion. Received: 10 July 1998 / Accepted: 19 October 1998