Intrinsic Capability of Budding Yeast Cofilin to Promote Turnover of Tropomyosin-Bound Actin Filaments

The ability of actin filaments to function in cell morphogenesis and motility is closely coupled to their dynamic properties. Yeast cells contain two prominent actin structures, cables and patches, both of which are rapidly assembled and disassembled. Although genetic studies have shown that rapid actin turnover in patches and cables depends on cofilin, how cofilin might control cable disassembly remains unclear, because tropomyosin, a component of actin cables, is thought to protect actin filaments against the depolymerizing activity of ADF/cofilin. We have identified cofilin as a yeast tropomyosin (Tpm1) binding protein through Tpm1 affinity column and mass spectrometry. Using a variety of assays, we show that yeast cofilin can efficiently depolymerize and sever yeast actin filaments decorated with either Tpm1 or mouse tropomyosins TM1 and TM4. Our results suggest that yeast cofilin has the intrinsic ability to promote actin cable turnover, and that the severing activity may rely on its ability to bind Tpm1

Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry

YesBackground. The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings. To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions. This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.This work was supported by the grants from the Flanders Research Foundation, SOFI-B Grant to CRK, http://www.fwo.be/, a Public Health Service Grant from the National Institutes of Health to CEC, (grant # AI-051334), https://www.nih.gov/ and a grant from the Grant Agency of the Czech Republic to DS and MS (P302/12/0574, GP14-29596P), https:// gacr.cz/

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed