Identification of an amino acid residue in the protein kinase C C1b domain crucial for its localization to the Golgi network

Protein kinase C (PKC) isoforms have been reported to be targeted to the Golgi complex via their C1 domains. We have shown recently that the regulatory domain of PKC induces apoptosis in neuroblastoma cells and that this effect is correlated to Golgi localization via the C1b domain. This study was designed to identify specific residues in the C1 domains that mediate Golgi localization. We demonstrate that the isolated C1b domains from PKCalpha, -delta, -epsilon, -eta, and - are targeted to the Golgi complex, whereas the corresponding C1a domains localize throughout the cell. Sequence alignment showed that amino acid residues corresponding to Glu-246 and Met-267 in PKC are conserved among C1b but absent from C1a domains. Mutation of Met-267, but not of Glu-246, to glycine abolished the Golgi localization of the isolated C1b domain and the regulatory domain of PKC. The mutated PKC regulatory domain constructs lacking Golgi localization were unable to induce apoptosis, suggesting a direct correlation between Golgi localization and apoptotic activity of PKC regulatory domain. Mutation of analogous residues in the C1b domain of PKCepsilon abrogated its Golgi localization, demonstrating that this effect is not restricted to one PKC isoform. The abolished Golgi localization did not affect neurite induction by PKCepsilon. However, the PKCepsilon mutant did not relocate to the Golgi network in response to ceramide and ceramide did not suppress the neurite-inducing capacity of the protein. Thus, the specific mutations in the C1b domain influence both the localization and function of full-length PKCepsilon.     

Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell surface and that catalytic activity of PKCepsilon is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 microM phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCepsilon could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKCepsilon both contain a binding site for ADAM12. These studies show that PKCepsilon plays a critical role in the regulation of ADAM12 cell-surface expression.     

Contest outcome in a territorial butterfly: the role of motivation

In many butterfly species, males compete over areas advantageous for encountering females. Rules for contest settlement are, however, largely unknown and neither morphological nor physiological traits can reliably predict the contest outcome. Here, we test the hypothesis that contests are settled in accordance with a motivation asymmetry. We staged contests between males of Pararge aegeria and after removing the resident, the non-resident was allowed (i) either to interact with a non-receptive female for 30 min (n = 30) or (ii) to spend 30 min alone in the cage (n = 30), after which the initial resident was reintroduced. The results show that males that had interacted with a female had a higher probability of becoming dominant and reversing contest outcome. Moreover, males that were faster to take over a vacant territory when the resident was removed were more likely to become dominant. Here, we show for the first time, to our knowledge, that frequent encounters with a mated female can increase male motivation to persist in a territorial contest in a butterfly. Further, we suggest that variation in intrinsic motivation reflects male eagerness to take over vacant territory. This study indicates that variation in resource value and motivational asymmetries are important for settling contests in butterflies.     

The river domain: why are there more species halfway up the river?

Biologists have long noted higher levels of species diversity in the longitudinal middle‐courses of river systems and have proposed many explanations. As a new explanation for this widespread pattern, we suggest that many middle‐course peaks in richness may be, at least in part, a consequence of geometric constraints on the location of species’ ranges along river courses, considering river headwaters and mouths as boundaries for the taxa considered. We demonstrate this extension of the mid‐domain effect (MDE) to river systems for riparian plants along two rivers in Sweden, where a previous study found a middle‐course peak in richness of natural (non‐ruderal) species. We compare patterns of empirical richness of these species to null model predictions of species richness along the two river systems and to spatial patterns for six environmental variables (channel width, substrate fineness, substrate heterogeneity, ice scour, bank height, and bank area). In addition, we examine the independent prediction of mid‐domain effects models that species with large ranges, because the location of their ranges is more constrained, are more likely to produce a mid‐domain peak in richness than are species with small ranges. Species richness patterns of riparian plants were best predicted by models including both null model predictions and environmental variables. When species were divided into large‐ranged and small‐ranged groups, the mid‐domain effect was more prominent and the null model predictions were a better fit to the empirical richness patterns of large‐ranged species than those of small‐ranged species. Our results suggest that the peak in riparian plant species richness in the middle courses of the rivers studied can be explained by an underlying mid‐domain effect (driven by geometric constraints on large‐ranged species), together with environmental effects on richness patterns (particularly on small‐ranged species). We suggest that the mid‐domain effect may help to explain similar middle‐course richness peaks along other rivers.     

A unique recent origin of the allotetraploid species Arabidopsis suecica: Evidence from nuclear DNA markers

A coalescent-based method was used to investigate the origins of the allotetraploid Arabidopsis suecica, using 52 nuclear microsatellite loci typed in eight individuals of A. suecica and 14 individuals of its maternal parent Arabidopsis thaliana, and four short fragments of genomic DNA sequenced in a sample of four individuals of A. suecica and in both its parental species A. thaliana and Arabidopsis arenosa. All loci were variable in A. thaliana but only 24 of the 52 microsatellite loci and none of the four sequence fragments were variable in A. suecica. We explore a number of possible evolutionary scenarios for A. suecica and conclude that it is likely that A. suecica has a recent, unique origin between 12,000 and 300,000 years ago. The time estimates depend strongly on what is assumed about population growth and rates of mutation. When combined with what is known about the history of glaciations, our results suggest that A. suecica originated south of its present distribution in Sweden and Finland and then migrated north, perhaps in the wake of the retreating ice.     

Developmental regulation of a VEIDase caspase-like proteolytic activity in barley caryopsis

Caspases are essential in animal programmed cell death both as initiator and executioner proteases. Plants do not have close caspase homologues, but several instances of caspase-like proteolytic activity have been demonstrated in connection with programmed cell death in plants. It was asked if caspase-like proteases are involved during development of the barley caryopsis. The presence of a caspase-6-like proteolytic activity that preferentially cleaved the sequence VEID was demonstrated. A range of protease inhibitors was tested and only caspase-specific inhibitors showed major inhibitory effects. The profile of VEIDase activity in developing starchy endosperm, embryo, and whole caryopsis was measured and showed a general trend of higher activity in young, rapidly developing tissues. The VEIDase activity was localized in vivo to vesicles, shown to be autophagosomes, in randomly distributed cells of the starchy endosperm. The VEIDase activity detected in barley caryopsis is similar to activities described previously in mammals, spruce, yeast, and thale cress. In mammals, spruce, and yeast, VEIDase activity has been shown to be positively correlated with the occurrence of programmed cell death. Several manifestations of programmed cell death exist in developing barley caryopsis, indicating a connection between VEIDase activity and developmental programmed cell death in barley.     

The effect of lactic acid on anaerobic carbon or nitrogen limited chemostat cultures of Saccharomyces cerevisiae

Weak organic acids are well-known metabolic effectors in yeast and other micro-organisms. High concentrations of lactic acid due to infection of lactic acid bacteria often occurs in combination with growth under nutrient-limiting conditions in industrial yeast fermentations. The effects of lactic acid on growth and product formation of Saccharomyces cerevisiae were studied, with cells growing under carbon- or nitrogen-limiting conditions in anaerobic chemostat cultures (D=0.1 h⁻¹) at pH values 3.25 and 5. It was shown that lactic acid in industrially relevant concentrations had a rather limited effect on the metabolism of S. cerevisiae. However, there was an effect on the energetic status of the cells, i.e. lactic acid addition provoked a reduction in the adenosine triphosphate (ATP) content of the cells. The decrease in ATP was not accompanied by a significant increase in the adenosine monophosphate levels.     

Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5'-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (oligochaeta) from Bermuda and the Bahamas

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5'-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.