Dataset Information

Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery

ABSTRACT: The recent identification of catalytically active peptidylglycine -amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). Homologs of prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) were identified in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectroscopy to identify proteins recovered from the soluble secretome of mating gametes and searched for evidence that the putative peptidergic processing enzymes were functional.After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact or were subjected to cleavage were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the soluble mating secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites and had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Navin Rauniyar

LAB HEAD: Betty A. Eipper

PROVIDER: PXD010945 | Pride | 2019-07-30

REPOSITORIES: Pride

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Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery.

Luxmi Raj R Blaby-Haas Crysten C Kumar Dhivya D Rauniyar Navin N King Stephen M SM Mains Richard E RE Eipper Betty A BA

Proteomes 20180923 4

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sex ...[more]

PMID: 30249063

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Project description:Chemostat experiments: Predator-prey dynamics were followed in four replicate chemostats (continuous-flow aquatic culture vessels) supplied with nitrogen-limited sterile medium that was otherwise sufficient in all nutrient constituents (Yoshida et al. 2003; Becks et al. 2010). Three chemostats contained 380 mL of medium, while the fourth, used for our algal gene expression study, contained 3000 mL so as to provide sufficient material for genetic analysis. Brachionus calyciflorus was isolated originally from Milwaukee harbor, Wisconsin, and provided by M. Boraas. Chlamydomonas reinhardtii was obtained from the University of Texas algal culture collection (UTEX 89). Both species reproduced asexually in our chemostat system. Though B. calyciflorus is naturally cyclically parthenogenetic, the rotifers in our stock culture have evolved not to produce males because the bubbling in our chemostats prevents mating; we used only one mating type of Chlamydomonas. Absence of sexual reproduction does not preclude the processes of microevolution (Bell 2009) that we study, and it is commonly accepted that asexual species can evolve (Lenski et al. 1991; Barrett et al. 2005). We used a Chlamydomonas lineage, founded from our original Chlamydomonas stock and then exposed continuously to Brachionus predation for six months in chemostat culture prior to this study during which Chlamydomonas evolved to form clumps (the 'grazed Chlamydomonas' lineage in Becks et al. 2010). We tracked changes in Chlamydomonas gene expression throughout two complete cycles of algal and rotifer abundance and concurrent cycles in algal defense. Algal samples were taken from one chemostat at intervals of approximately every ten days (8 dates total), chosen to capture critical transitions in cell clumping. A sample taken at the start of the experiment from the culture that was used to inoculate the chemostat was the standard against which algae from later samples were compared (i.e., the M-^Sgrazed ChlamydomonasM-^T). To obtain sufficient material for microarray analysis, ca. 1 - 1.5 L algal samples were collected and filtered through a 10-um mesh to exclude rotifers and rotifer eggs. The algal samples of ca. 100 ml of chemostat medium were concentrated by centrifugation and frozen. Samples were shock frozen in liquid nitrogen and transferred to -80C.

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Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery

Publications

Proteases Shape the <i>Chlamydomonas</i> Secretome: Comparison to Classical Neuropeptide Processing Machinery.

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