Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.

Oil bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward deciphering oil accumulation mechanisms in algae, we used proteomics to analyze purified oil bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (≥ 2 peptides) identified by LC-MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl-lipids and sterols). Compared with a recently reported Chlamydomonas oil body proteome, 19 new proteins of lipid metabolism were identified, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol-3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated with the oil body fraction. This data set thus provides evidence that Chlamydomonas oil bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil bodies in microalgal cell functions.

Deinococcaceae are a family of extremely radiation-tolerant bacteria that are currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. To achieve a comprehensive and accurate annotation of the Deinococcus deserti genome, we performed an N terminus-oriented characterization of its proteome. For this, we used a labeling reagent, N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide, to selectively derivatize protein N termini. The large scale identification of N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide-modified N-terminal-most peptides by shotgun liquid chromatography-tandem mass spectrometry analysis led to the validation of 278 and the correction of 73 translation initiation codons in the D. deserti genome. In addition, four new genes were detected, three located on the main chromosome and one on plasmid P3. We also analyzed signal peptide cleavages on a genome-wide scale. Based on comparative proteogenomics analysis, we propose a set of 137 corrections to improve Deinococcus radiodurans and Deinococcus geothermalis gene annotations. Some of these corrections affect important genes involved in DNA repair mechanisms such as polA, ligA, and ddrB. Surprisingly, experimental evidences were obtained indicating that DnaA (the protein involved in the DNA replication initiation process) and RpsL (the S12 ribosomal conserved protein) translation is initiated in Deinococcaceae from non-canonical codons (ATC and CTG, respectively). Such use may be the basis of specific regulation mechanisms affecting replication and translation. We also report the use of non-conventional translation initiation codons for two other genes: Deide_03051 and infC. Whether such use of non-canonical translation initiation codons is much more frequent than for other previously reported bacterial phyla or restricted to Deinococcaceae remains to be investigated. Our results demonstrate that predicting translation initiation codons is still difficult for some bacteria and that proteomics-based refinement of genome annotations may be helpful in such cases.

Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent processive metalloprotease FtsH and cleaves MgtE, the major highaffinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn 2+ /Zn 2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn 2+ and Zn 2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of "ERAD" in bacteria and exemplifies an ancestral function of rhomboid-superfamily proteins.

In skeletal muscle, the triad is a structure comprising a transverse (T)-tubule and sarcoplasmic reticulum (SR) cisternae. Triads constitute the basis of excitation-contraction coupling as the cradle of the Ca 2+ release complex. We have shown previously that triadin, a member of this complex, has shaping properties on reticulum membrane and is indirectly involved in a link between triads and microtubules. We have identified here that CLIMP-63 (also known as CKAP4), as the partner of triadin, is responsible for this association of triads and microtubules. Triadin and CLIMP-63 interact through their respective luminal domains and the shaping properties of triadin depend on the capacity of CLIMP-63 to bind microtubules with its cytosolic portion. In skeletal muscle, CLIMP-63 is localized in the SR, including triads, and is associated with the Ca 2+ release complex through its interaction with triadin. Knockout of triadin in muscles results in the delocalization of CLIMP-63 from triads, its dissociation from the Ca 2+ release complex and a disorganization of the microtubule network. Our results suggest that the association of triadin and CLIMP-63 could be involved in the shaping of SR terminal cisternae and in the guidance of microtubules close to the triads.

Ca(2+)/Calmodulin (CaM)-dependent signaling pathways play a major role in the modulation of cell responses in eukaryotes. In the chloroplast, few proteins such as the NAD(+) kinase 2 have been previously shown to interact with CaM, but a general picture of the role of Ca(2+)/CaM signaling in this organelle is still lacking. Using CaM-affinity chromatography and mass spectrometry, we identified 210 candidate CaM-binding proteins from different Arabidopsis and spinach chloroplast sub-fractions. A subset of these proteins was validated by an optimized in vitro CaM-binding assay. In addition, we designed two fluorescence anisotropy assays to quantitatively characterize the binding parameters and applied those assays to NAD(+) kinase 2 and selected candidate proteins. On the basis of our results, there might be many more plastidial CaM-binding proteins than previously estimated. In addition, we showed that an array of complementary biochemical techniques is necessary in order to characterize the mode of interaction of candidate proteins with CaM.

Supplementary data: http://dx.doi.org/10.1016/j.neps.2016.08.001International audienc

The eukaryotic cap-binding complex (CBC) is a hub for regulations affecting mRNA behaviour including translation, degradation and storage. Beside the core eukaryotic translation initiation factors, other proteins, many of which are yet unknown, are thought to interact stably or transiently with the CBC depending on cell status. The prototype of these regulators is the animal eIF4E binding protein (4E-BP), a direct target of the TOR (Target of Rapamycin) kinase that competes with the cap-binding protein eIF4E, thus repressing translation. In plants, no functional homologs of 4E-BP have so far been characterized. In this work we performed several deep proteomic analyses of the Arabidopsis CBC after cap-affinity purification from wild-type plants. We also investigated the CBC in eIF4E mutant plants, Arabidopsis lines with lower TOR activity, or during infection with eIF4E-dependent potyviruses, conditions which are all affecting translation at the initiation level. These analyses allowed us to define a limited core set of CBC components, which were detected in all samples. Interestingly, we identified proteins, like AGO1 or VCS, which were always detected in conditions where either TOR or mRNA translation were reduced. Meta-analysis of these data revealed several new plant interactors of the CBC, potentially defining pathways related to mRNA stability and degradation, metabolism and viral life cycle. A search for eIF4E binding motifs identified several new potential 4E-BP relatives in plants.