Biochemical studies on lysin, a cell wall degrading enzyme released during fertilization in Chlamydomonas

Buchanan, M. J.; Snell, William J.

doi:10.1016/0014-4827(88)90357-6

Cited by 32 publications

(27 citation statements)

References 32 publications

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“…To examine the requirement for gamete activation, we mixed unactivated, SYTOX-labeled, glutaraldehyde-fixed, wild-type mtϩ gametes with activated, live imp12 mtϪ gametes and assessed pair formation by using the docking assay. Before mixing, we removed the extracellular matrix (cell wall) that encloses unactivated gametes by use of the wall-degrading Chlamydomonas collagenase, g-lysin (Buchanan and Snell, 1988;Kinoshita et al, 1992). We found that only activated gametes were capable of docking (Figure 3).…”

Section: Gamete Activation and A Functional Fus1 Gene Are Essential Fmentioning

confidence: 99%

TheChlamydomonasFus1 Protein Is Present on the Mating TypeplusFusion Organelle and Required for a Critical Membrane Adhesion Event during Fusion withminusGametes

Misamore

Gupta

Snell³

2003

MBoC

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The molecular mechanisms of the defining event in fertilization, gamete fusion, remain poorly understood. The FUS1 gene in the unicellular, biflagellated green alga Chlamydomonas is one of the few sex-specific eukaryotic genes shown by genetic analysis to be essential for gamete fusion during fertilization. In Chlamydomonas, adhesion and fusion of the plasma membranes of activated mtϩ and mtϪ gametes is accomplished via specialized fusion organelles called mating structures. Herein, we identify the endogenous Fus1 protein, test the idea that Fus1 is at the site of fusion, and identify the step in fusion that requires Fus1. Our results show that Fus1 is a ϳ95-kDa protein present on the external surface of both unactivated and activated mtϩ gametes. Bioassays indicate that adhesion between mating type plus and mating type minus fusion organelles requires Fus1 and that Fus1 is functional only after gamete activation. Finally, immunofluorescence demonstrates that the Fus1 protein is present as an apical patch on unactivated gametes and redistributes during gamete activation over the entire surface of the microvillous-like activated plus mating structure, the fertilization tubule. Thus, Fus1 is present on mtϩ gametes at the site of cell-cell fusion and essential for an early step in the fusion process. INTRODUCTIONGamete fusion defines the beginning of a new organism in all sexual species. In most organisms, the events that precede fusion have been well characterized at the cellular level (Snell and White, 1996;Wassarman et al., 2001;Primakoff and Myles, 2002). Initial cell-cell interactions between sperm surface proteins and extracellular matrix molecules on the egg trigger activation of the sperm and exposure of previously cryptic regions of the plasma membrane proposed to be involved in gamete fusion. Once the sperm has made its way to the egg plasma membrane, the membranes of the two gametes interact more intimately, finally bringing about gamete fusion. Several sperm and egg molecules implicated in activation of the sperm have been identified, and we know much about the morphological events that accompany gamete fusion (Yanagimachi, 1994). On the other hand, little is known about the molecular mechanisms that underlie the late steps in fertilization, when the plasma membranes of the interacting gametes adhere and fuse (for review, see Primakoff and Myles, 2002). In the absence of any purely eukaryotic systems in which bona fide fusion proteins have been identified, the best models for fusion of the external plasma membrane of cells have come from studies of fusion of viruses with their eukaryotic target cells (White, 1996;Eckert and Kim, 2001). In these systems, a viral transmembrane surface protein interacts with a receptor in the host cell plasma membrane, leading to docking of the virus particle on the cell surface. Subsequent conformational changes in the interacting proteins finally lead to complete fusion (Dimitrov, 2000;Malashkevich et al., 2001;Mayer, 2001).A small number of eukaryotic genes has been sho...

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Section: Gamete Activation and A Functional Fus1 Gene Are Essential Fmentioning

confidence: 99%

TheChlamydomonasFus1 Protein Is Present on the Mating TypeplusFusion Organelle and Required for a Critical Membrane Adhesion Event during Fusion withminusGametes

Misamore

Gupta

Snell³

2003

MBoC

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“…Methods for cell culture were essentially as previously described (5). Gametogenesis was induced by transferring cells grown in acetate-supplemented,medium to nitrogen-free medium diluted to 3/4 strength, and aerating in continuous light for 14-20 h. The resulting gametes were harvested by allowing them to accumulate at the bottom of the bottles by negative phototaxis/positive geotaxis (4) without aeration, and the supematant was removed by siphoning.…”

Section: Cell Culture and Preparation Of Gametesmentioning

confidence: 99%

“…p-Lysinase was assayed for by its ability to generate lysin from prolysin either by use of immunoblotting as described above or by a wall loss assay (5). To use the wall loss assay for p-lysinase, the sample containing p-lysinase 1.…”

Section: Assaying For P-lysinasementioning

confidence: 99%

“…or buffer were incubated with supernatants from nonmating or mating, de-walled gametes and then assayed in a wall loss assay (5). The results shown in Fig.…”

Section: Prolysin Converting Activity Is Released Into the Medium Durmentioning

confidence: 99%

“…Our laboratory has been interested in understanding the molecular details of activation of lysin, which is one of the earliest of these signaled events. It is known, from work by us and others (5,12,14), that the lysin released by mating gametes is a 60,000-Mr metalloprotease and recently we reported that it acts on a flagellar collar molecule as well as the highly cross-linked framework of the cellulose-deficient, glycoprotein-rich cell wall of Chlamydomonas (11). Other workers had presented evidence consistent with the idea that lysin was in the periplasm of gametes (18) and Matsuda et al (15) reported that lysin could be isolated from homogenates of gametes.…”

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Regulated secretion of a serine protease that activates an extracellular matrix-degrading metalloprotease during fertilization in Chlamydomonas.

Snell

Eskue

Buchanan

1989

The Journal of Cell Biology

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Abstract. During fertilization in the biflagellated alga, Chlamydomonas reinhardtii, gametes of opposite mating types adhere to each other via agglutinin molecules located on their flagellar surfaces, generating a sexual signal that induces several cellular responses including cell wall release. This cell contact-generated signal is mediated by cAMP and release of the wall, which is devoid of cellulose and contains several hydroxyproline-rich glycoproteins, is due to the activation of a metalloprotease, lysin. Although we originally assumed that lysin would be stored intracellulady in a compartment structurally separate from its substrate, recently we showed that lysin is stored in the periplasm as an inactive, higher relative molecular mass precursor, prolysin (Buchanan, M. J., S. H.

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Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes

1995

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Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization.

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Biochemical studies on lysin, a cell wall degrading enzyme released during fertilization in Chlamydomonas

Cited by 32 publications

References 32 publications

TheChlamydomonasFus1 Protein Is Present on the Mating TypeplusFusion Organelle and Required for a Critical Membrane Adhesion Event during Fusion withminusGametes

TheChlamydomonasFus1 Protein Is Present on the Mating TypeplusFusion Organelle and Required for a Critical Membrane Adhesion Event during Fusion withminusGametes

Regulated secretion of a serine protease that activates an extracellular matrix-degrading metalloprotease during fertilization in Chlamydomonas.

Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes

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