N-Glycosylation in Piroplasmids: Diversity within Simplicity

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins; Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.

Stress responses of the oil-producing green microalgaBotryococcus brauniiRace B

Plants react to biotic and abiotic stresses with a variety of responses including the production of reactive oxygen species (ROS), which may result in programmed cell death (PCD). The mechanisms underlying ROS production and PCD have not been well studied in microalgae. Here, we analyzed ROS accumulation, biomass accumulation, and hydrocarbon production in the colony-forming green microalgaBotryococcus brauniiin response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. We also identified and cloned a single cDNA for theB. brauniiortholog of theArabidopsisgenedefender against cell death 1(DAD1),a gene that is directly involved in PCD regulation. The function ofB. braunii DAD1was assessed by a complementation assay of the yeast knockout line of theDAD1ortholog, oligosaccharyl transferase 2. Additionally, we found thatDAD1transcription was induced in response to SA at short times. These results suggest thatB. brauniiresponds to stresses by mechanisms similar to those in land plants and other organisms.

Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly

Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation.