The transcriptome of Candida albicans mitochondria and the evolution of organellar transcription units in yeasts.

BACKGROUND Yeasts show remarkable variation in the organization of their mitochondrial genomes, yet there is little experimental data on organellar gene expression outside few model species. Candida albicans is interesting as a human pathogen, and as a representative of a clade that is distant from the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Unlike them, it encodes seven Complex I subunits in its mtDNA. No experimental data regarding organellar expression were available prior to this study. METHODS We used high-throughput RNA sequencing and traditional RNA biology techniques to study the mitochondrial transcriptome of C. albicans strains BWP17 and SN148. RESULTS The 14 protein-coding genes, two ribosomal RNA genes, and 24 tRNA genes are expressed as eight primary polycistronic transcription units. We also found transcriptional activity in the noncoding regions, and antisense transcripts that could be a part of a regulatory mechanism. The promoter sequence is a variant of the nonanucleotide identified in other yeast mtDNAs, but some of the active promoters show significant departures from the consensus. The primary transcripts are processed by a tRNA punctuation mechanism into the monocistronic and bicistronic mature RNAs. The steady state levels of various mature transcripts exhibit large differences that are a result of posttranscriptional regulation. Transcriptome analysis allowed to precisely annotate the positions of introns in the RNL (2), COB (2) and COX1 (4) genes, as well as to refine the annotation of tRNAs and rRNAs. Comparative study of the mitochondrial genome organization in various Candida species indicates that they undergo shuffling in blocks usually containing 2-3 genes, and that their arrangement in primary transcripts is not conserved. tRNA genes with their associated promoters, as well as GC-rich sequence elements play an important role in these evolutionary events. CONCLUSIONS The main evolutionary force shaping the mitochondrial genomes of yeasts is the frequent recombination, constantly breaking apart and joining genes into novel primary transcription units. The mitochondrial transcription units are constantly rearranged in evolution shaping the features of gene expression, such as the presence of secondary promoter sites that are inactive, or act as "booster" promoters, simplified transcriptional regulation and reliance on posttranscriptional mechanisms

The role of hydrophobic interactions in ankyrin–spectrin complex formation

AbstractSpectrin and ankyrin are the key components of the erythrocyte cytoskeleton. The recently published crystal structure of the spectrin–ankyrin complex has indicated that their binding involves complementary charge interactions as well as hydrophobic interactions. However, only the former is supported by biochemical evidence. We now show that nonpolar interactions are important for high affinity complex formation, excluding the possibility that the binding is exclusively mediated by association of distinctly charged surfaces. Along these lines we report that substitution of a single hydrophobic residue, F917S in ankyrin, disrupts the structure of the binding site and leads to complete loss of spectrin affinity. Finally, we present data showing that minimal ankyrin binding site in spectrin is formed by helix 14C together with the loop between helices 15 B/C

Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton

Expression, purification and functional characterization of recombinant human acyl-CoA-binding protein (ACBP) from erythroid cells

Fatty acyl-CoA esters are extremely important in cellular homeostasis. They are intermediates in both lipid metabolism and post-translational protein modifications. Among these modification events, protein palmitoylation seems to be unique by its reversibility which allows dynamic regulation of the protein hydrophobicity. The recent discovery of an enzyme family that catalyze protein palmitoylation has increased the understanding of the enzymology of the covalent attachment of fatty acids to proteins. Despite that, the molecular mechanism of supplying acyl-CoA esters to this reaction is yet to be established. Acyl-coenzyme A-binding proteins are known to bind long-chain acyl-CoA esters with very high affinity. Therefore, they play a significant role in intracellular acyl-CoA transport and pool formation. The purpose of this work is to explore the potential of one of the acyl-CoA-binding proteins to participate in the protein palmitoylation. In this study, a recombinant form of ACBP derived from human erythroid cells was expressed in E. coli, purified, and functionally characterized. We demonstrate that recombinant hACBP effectively binds palmitoyl-CoA in vitro, undergoing a shift from a monomeric to a dimeric state, and that this ligand-binding ability is involved in erythrocytic membrane phosphatidylcholine (PC) remodeling but not in protein acylation

Pedagogy as possibility: Health interventions as digital openness

In this article we propose an approach to digital health tracking technologies that draws on design anthropology. This entails re-thinking the pedagogical importance of personal data as lying in how they participate in the constitution of new possibilities that enable people to learn about, and configure, their everyday health in new ways. There have been two dominant strands in traditional debates in the field of pedagogy: one that refers to processes of teaching people to do things in particular ways; and another that seeks to enable learning. The first of these corresponds with existing understandings of self-tracking technologies as either unsuccessful behavioural change devices, or as providing solutions to problems that do not necessarily exist. When seen as such, self-tracking technologies inevitably fail as forms of intervention towards better health. In this article we investigate what happens when we take the second strand-the notion of enabling learning as an incremental and emergent process-seriously as a mode of intervention towards health through self-tracking technologies. We show how such a shift in pedagogical understanding of the routes to knowing these technologies offer creates opportunities to move beyond simplistic ideas of behavioural change as the main application of digital body monitoring in everyday life. In what follows, we first demonstrate how the disjunctures that arise from this context emerge. We then outline a critical response to how learning through life-tracking has been conceptualised in research in health and human-computer interaction research. We offer an alternative response by drawing on a processual theory of learning and recent and emerging research in sociology, media studies, anthropology, and cognate disciplines. Then, drawing on ethnographic research, we argue for understanding learning through the production of personal data as involving emplaced and non-representational routes to knowing. Thi

Additional file 6: Table S6. of The transcriptome of Candida albicans mitochondria and the evolution of organellar transcription units in yeasts

Mitochondrial genome sequences used in the evolutionary comparisons. Genome size, accession numbers, number of introns, and references are provided. (XLSX 14 kb

Additional file 2: Table S2. of The transcriptome of Candida albicans mitochondria and the evolution of organellar transcription units in yeasts

Promoters and promoter-like sequences in C. albicans mtDNA. Promoters of the primary transcription units are highlighted. In the “support” column “none” means that a consensus nonanucleotide is present, but no transcriptional activity can be reliably attributed to the promoter, “reads” means that there is an increased number of reads mapping downstream of the promoter, “reads and 5’-RACE” means promoters confirmed independently (Fig. 4). The second copy of inverted repeat (IRb) is omitted, as in Fig. 2b. (XLSX 9 kb

Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of beta I-Spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that "opening" of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton