Light dependence of sexual agglutinability in Chlamydomonas eugametos

The mating activity of mating-type plus gametes of Chlamydomonas eugametos depends on light. Cells lost their ability to agglutinate with mating-type minus gametes after a dark period of 30 min. They regained their agglutinability after 10 min exposure to light. Other mating reactions, such as tipping and flagellar tip activation, were not dependent upon light. Since cycloheximide and tunicamycin did not affect the light-induced activation of flagellar agglutinability, no protein synthesis or glycosylation is involved in this process. Equal amounts of biologically active agglutination factor could be extracted from cells placed either in light or in darkness. A minor portion of the active material was found to be located on the flagellar surface of illuminated cells. No active material was found on the flagellar surface of dark-exposed cells, whereas their cell bodies contained the same amount of active material as the cell bodies of illuminated cells. Since a light-induced flow of agglutination factors from the cell body to the flagella could not be detected and dark-exposed cells could be slightly activated by amputation or fixation by glutaraldehyde, we propose that light affects flagellar agglutinability by an in-situ modification of the agglutination factor on the flagella. When mt ⁺ and mt ^- strains were crossed and the progeny examined for light-sensitivity, it was apparent that this phenomenon is not mating type-linked.Abbreviations and symbols FTA flagellar tip activation - mt ^+/- mating type plus or minus - WGA wheat-germ agglutinin     

Sex-specific binding and inactivation of agglutination factor in Chlamydomonas eugametos

Gametes of opposite mating type (mt ⁺ and mt ^-) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using ³⁵S-labeled flagella and the isolated mt ^-agglutination factor. It is shown that not only isolated flagella, but also the mt ^-agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt ^-agglutination factor in determining the sexual agglutinability of mt ^-gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt ^-agglutination factor can be completely inactivated.Abbreviations Mt ^+/- mating type plus or minus - PAS periodic-acid Schiff-reagent - Hepes 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - HMC buffer Hepes buffer (10 mM. pH 7.2, containing 1 mM MgCl₂ and 1 mM CaCl₂)     

Two tunicamycin-sensitive components involved in agglutination and fusion ofChlamydomonas reinhardtii gametes

To find out glycoproteins involved in the mating reaction ofChlamydomonas reinhardtii, the effect of tunicamycin (TM), a potent inhibitor of glycosylation of proteins, was studied. TM, when present during gametogenesis, blocked the acquisition of agglutinability ofmt ⁺ cells. TM also inhibited the recovery of agglutinability ofmt ⁺ gamete after trypsin treatment. On the contrary, TM blocked neither the acquisition of agglutination during gametogenesis ofmt ^- cells nor the recovery of their agglutinability after trypsinization. It was found, however, that the TM-treatedmt ^- gametes can agglutinate but do not fuse with non treatedmt ⁺ gametes at all. When gametes of gam-1mt ^-, a conditional mutant strain for cell fusion, were induced at non permissive temperature of 35°C and then transferred to 25°C, the ability of cell fusion was acquired after about 5 h incubation. Presence of TM completely blocked this acquisition. Based on these evidence, we conclude that at least two TM-sensitive glycoproteins are included in the mating reaction. The first component is located on the flagellar surface ofmt ⁺ gamete and responsible for agglutination withmt ^- flagella. The second component occurs on the surface ofmt ^- gamete and plays a role in the fusion withmt ⁺ gamete.Abbreviations CHI cycloheximide - mt mating type - TM tunicamycin     

Synthesis and turnover of cell body-agglutinin as a pool of flagellar surface-agglutinin inChlamydomonas reinhardii gamete

Chlamydomonas reinhardii cells were broken in a French press and the soluble fraction was tested for agglutination activity. Deflagellated cell bodies ofmt ⁺ andmt ^- gametes yielded soluble fractions that were able to isoagglutinate gametes of the opposite mating type. When the wild-type gametes of opposite mating types were mixed, the cell body-agglutinins were used up during flagellar agglutination and subsequent cell fusion. When thefus mt ⁺ andmt ^- gametes agglutinated without successive fusion, the amount of cell body-agglutinins sharply decreased, then increased and reached the premixing level: the recovery was blocked by cycloheximide. When cells were treated with EDTA or trypsin, the cell body-agglutinins as well as flagellar surface-agglutinins were completely lost without apparent loss of motility. The EDTA extract contained the same amount of agglutinins as observed in the cell bodies before extraction, and this amount was about 100 times higher than that in the EDTA extract of isolated flagella. By the addition of trypsin inhibitor, the trypsinized gametes resynthesized the cell body-agglutinins. The process was sensitive to cycloheximide in both mating type gametes and to tunicamycin inmt ⁺ gametes.Abbreviations mt ^+/- mating type plus or minus - CHI cycloheximide - TI trypsin inhibitor - TM tunicamycin     

An antiserum against a glycoprotein functional in flagellar adhesion between Chlamydomonas eugametos gametes

Chlamydomonas eugametos gametes agglutinate sexually by their flagellar surfaces. The agglutination factor on mating type minus (mt^-) gametes is thought to be a glycoprotein named PAS-1.2. To test this idea, an antiserum was raised against purified PAS-1.2., which reacted with isolated PAS-1.2 (immunoprecipitation tests) and blocked the ability of isolated PAS-1.2 to induce sexual twitching in mt ⁺ gametes. When tested with living cells, the antiserum specifically agglutinated mt ^- gametes and induced a reaction resembling twitching. Mt ⁺ flagella were shown to bind the antiserum (indirect immunofluorescence) but much less than mt ^- gametes. Mt ^- gametes pretreated with Fab fragments of the antiserum were unable to reproduce sexually, while treated mt ⁺ gametes were unaffected. This effect presumably results from the ability of the serum to block mt ^- sexual agglutination, for mt ^- isoagglutinin was completely inactivated by the serum, while mt ⁺ isoagglutinin was unaffected. It is therefore argued that PAS-1.2 is the in vivo mt ^- agglutination factor. However it is shown that the antiserum was able to react in vitro not only with PAS-1.2 but with several other proteins in both mt ^- and mt ⁺ flagella.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid-Schiff - GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PBS phosphate buffer-saline - Fab univalent antibody fragment The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)     

Reconstitution of biological activity in isoagglutinins fromChlamydomonas eugametos

Gametes ofChlamydomonas eugametos produce membrane vesicles, called isoagglutinins, which are shed into the culture fluid. It is assumed that they originate from the flagellar membrane for, like flagella, they can bind to the flagellar surface of gametes of the opposite mating type (mt). The composition ofmt ^- isoagglutinin was investigated with respect to this agglutinability. When the agglutination factor present on the surface ofmt ^- isoagglutinins (PAS-1.2) was removed, together with other membrane bound glycoproteins, the membrane vesicles were rendered inactive. They could be reactivated however by incubation with the extracted glycoproteins in a time-and concentration-dependent manner. The agglutination factor proved to be necessary yet sufficient in itself for the reactivation process to occur. Experiments with CsCl density gradients showed that the agglutination factor truly bound to the vesicles during reactivation. Inactivated vesicles derived frommt ⁺ gametes could be reactivated to gainmt ^- properties. Reactivation was inhibited by prior treatment with trypsin. The results indicate that the agglutination factor inmt ^- isoagglutinins is an extrinsic membrane protein bound to an intrinsic proteinaceous receptor.Abbreviations GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PAS periodic acid Schiff     

Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells

Sex-specific agglutinins from the cell surface of haploid cells of Saccharomyces cerevisiae (X2180, mt^a and mt) were purified and analysed. The constitutive agglutinin from mt^a cells was extractable with 3 mM dithiothreitol. It was shown to be a glycoprotein (3% mannose) with an apparent Mr of 43,000 based on gel filtration, but in SDS-PAGE it behaved as a much smaller molecule (Mr between 18,000 and 26,000). About one in three amino acids was a hydroxyamino acid. Its biological activity was resistant to boiling for 1 h, but sensitive to pronase. Intact mt cells retained their agglutinability in the presence of dithiothreitol but limited trypsinizing released a biologically active agglutinin fragment. It had an apparent Mr of 320,000 (gel filtration). When analysed by SDS-PAGE, a single diffuse band with an apparent Mr of 225,000 was observed. The protein was 94% (w/w) mannose with a trace of N-acetyl glucosamine. Its biological activity was almost completely lost after boiling for 1 h. Both agglutinins behaved as monovalent molecules and specifically inhibited the biological activity of both noninduced and pheromone-induced cells. Pheromone treatment of mt^a cells resulted in an apparent 32-fold increase in agglutinin activity at the cell surface, whereas pheromone treatment of mt cells only doubled the apparent agglutinin activity.Abbreviations mt mating type - DTT dithiothreitol - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - YPG yeast-peptone-glucose - PAS periodic-acid-Schiff reagent     

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt⁺ or mt^-). It is shown that before mt⁺ and mt^- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt^- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt⁺ gametes constituting the outer surface of the clumps with the mt^- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt⁺ flagellar tips then deactivate and swimming is resumed. The original mt^- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt^- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos

Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.Abbreviations db-cAMP dibutyryl-cAMP - FTA flagellar tip activation - Mab monoclonal antibody - mt ^–/mt⁺ mating-type minus/plus - WGA wheat-germ agglutinin We gratefully acknowledge the fruitful discussions with Dr. Rainer Gilles of the Department of Biochemistry at the University of Cologne (FRG), and the advice generously given by Dr. Roel van Driel of the Department of Biochemistry at the University of Amsterdam (The Netherlands).     

Transmission of mitochondrial DNA in crosses involving diploid gametes homozygous or heterozygous for the mating-type locus in Chlamydomonas

Summary The two interfertile algal species Chlamydomonas reinhardtii and C. smithii possess physically distinct mitochondrial (mit) genomes. Recently, use was made of this difference to demonstrate that sexual zygotes transmit the mit DNA from the mating-type minus (mt ^-, or paternal) parent exclusively. Diploid clones homozygous or heterozygous for the mt locus and carrying the mit genome of either of the two species were constructed by sexual crosses or artificially induced fusions. Haploid x diploid and diploid x diploid crosses were performed in order to analyze the role of both the mt locus and ploidy on the mode of transmission of mit DNA to the meiotic progeny. The inheritance of the mit DNA was determined by use of two molecular probes which hybridize to different regions of the organelle genomes. The mt ^u+/mt ^- gametes, which behave as mt ^- in the mating reaction, usually transmit their mit genome to the meiotic progeny, as do mt ^- or mt ^-/mt ^- gametes, regardless of the ploidy of the mt ⁺ gametes. In the cross mt ⁺ x mt ⁺/mt ^- however, 2 zygospore clones (out of 14) transmitted recombinant DNA molecules containing a large segment of the C. reinhardtii mit genome and a 1 kb fragment typical of C. smithii. It can thus be concluded that, contrary to what was observed earlier for chloroplast gene transmission: (1) mt ^- is dominant to mt ⁺with regard to mit DNA transmission, and (2) nuclear ploidy has little, if any, effect on mit DNA transmission.     

Sexual agglutinin of mating-type minus gametes inChlamydomonas reinhardtii

The sexual agglutinin from the mating-type minus gametes ofChlamydomonas reinhardtii was purified by gel filtration and hydroxyapatite chromotography. The minus agglutinin was identified as a single glycopolypeptide termed Agg(-) of very high molecular weight by SDS-poly-acrylamide gel electrophoresis. It was also observed as a glycoprotein with agglutinin activity on non-SDS polyacrylamide gels. The native agglutinin appeared to be composed of a complex of Agg(-) subunits. It consisted of about 60% protein and 40% carbohydrate and the activity was diminished by a mild periodate oxidation. When the plus agglutinin was also purified and compared with the minus agglutinin, it was found that both agglutinins migrate in the same position by SDS-polyacrylamide gel electrophoresis, whereas their behaviors on gel filtration and hydroxyapatite chromatography are different.Abbreviations mt ^+/– mating-tape plus or minus - SDS sodium dodecyl sulfate - V_e elution volume - V_o void volume - kDa kilodalton     

An inhibitor of cell fusion in Chlamydomonas eugametos

By a short treatment with acid of mt ^- gametes of Chlamydomonas eugametos, a factor is released which prevents gametic cell fusion, without affecting the viability of the cells. It has a very rapid action. By means of scanning electron microscopy it is shown that the factor has no influence on flagellar adhesion nor on the formation of a plasma papilla by cells of either mating type, but that it specifically inhibits the fusion of these papillae. Evidence is presented suggesting that this inhibitor has a predominant effect on mt ⁺ gametes. In cell pairs which are blocked with respect to papillar fusion, no flagellar disengagement occurs, which indicates that loss of agglutinability is a direct consequence of cell fusion.     

Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

Genetic Analysis of Mating Locus Linked Mutations in CHLAMYDOMONAS REINHARDII

The mating-type (mt) locus of Chlamydomonas reinhardii has been analyzed using four mutant strains (imp-1, imp-10, imp-11 and imp-12). All have been shown, or are shown here, to carry mutations linked to either the plus (mt⁺) or the minus (mt^-) locus, and their behavior in complementation tests has allowed us to define several distinct functions for each locus. Specifically, we propose that the mt⁺ locus contains the following genes or regulatory elements: a locus designated sfu, which is necessary for sexual fusion between gametes; a locus designated upp (uniparental plus), which controls aspects of chloroplast gene inheritance and perhaps also zygote maturation; and a locus designated sad, which functions in sexual adhesion. The mt^- locus also contains a sad locus as well as a gene or regulatory element designated mid, which is necessary for the minus dominance in mt⁺/mt^- diploids.     

Sexual agglutination factor from Chlamydomonas eugametos

Chlamydomonas eugametos gametes can sexually agglutinate via their flagellar surfaces whereas vegetative cells cannot. Therefore, flagellar glycoproteins, present in gamete cells but absent from vegetative cells, were investigated as prospective mt ^-agglutination factors. They were identified as periodic acid Schiff (PAS) stained bands separated in sodium dodecyl sulphate-polyacrylamide electrophoresis gels. Gamete-specific bands were determined by comparison with equivalent gels of vegetative flagella and by immunological techniques using antisera raised against isolated mt ^- gamete flagella. Four high molecular weight flagellar glycoproteins proved to be gamete specific (PAS-1.2, PAS-1.3, PAS-3 and PAS-4). They were extracted from flagella by 3 M guanidine thiocyanate, separated in a column of Sepharose 2B, and tested for in vitro agglutination activity on mt ⁺ gametes. A single peak of activity was found to be correlated with the presence of the PAS-1.2 band. It is shown that mt ^- agglutination activity is related to the concentration of this glycoprotein in flagellar membranes.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid Schiff - GTC guanidine thiocyanate - mt ^-/+ mating type plus or minus     

Light Affects Flagellar Agglutinability in Chlamydomonas eugametos by Modification of the Agglutinin Molecules

The effect of light on the sexual competence of a light-sensitive mating type minus strain (mt⁻) of Chlamydomonas eugametos obtained by crossing a light-sensitive mating type plus strain (mt⁺) with a light-insensitive mt⁻ strain is described. As previously demonstrated for the mt⁺ parent, this study of one of the mt⁻ offspring shows that (a) a light-sensitive mechanism affects flagellar agglutinability in a rapid process that does not require protein synthesis; (b) only the activity of the flagellar agglutinins (glycoproteins responsible for agglutination) is susceptible to light while agglutinins on the cell body surface are not affected by light. We further demonstrate that (a) membrane vesicles naturally released from nonagglutinable dark gametes remain inactive. Extracts of these vesicles also remain inactive even though they contain agglutinin-like components; (b) inactive mt⁻ agglutinin is present in extracts of flagella from nonagglutinable dark gametes by comparison of its chromatographic, electrophoretic, and immunogenic properties with those of active agglutinin. When purified of all other flagellar proteins, it remains inactive; (c) a monoclonal antibody directed against the sexual agglutination site of the mt⁻ agglutintin discriminates between active and inactive agglutinins when present in a native state on the flagellar surface, but is unable to discriminate between them when they are denatured in sodium dodecyl sulfate-electrophoresis gels and blotted onto nitrocellulose. Taken collectively these observations suggest that light activation involves the chemical modification of the agglutinins in situ on the flagellar surface.     

–SH and S–S involvement in Chlamydomonas mating

Reagents that block or cross-link sulfhydryl (–SH) groups and those that reduce disulfide (S–S) bonds have been tested for their effects on mating in Chlamydomonas reinhardii. Wild-type (wt) gametes of mating type + (mt⁺) and mt^?, and a fusion-defective mt^? mutant, gam-11, were studied. Differential sensitivities of mt⁺ vs mt^? and of wt mt^? vs gam-11 mt^? were analyzed. Concentrations of reagents that did not disrupt flagellar agglutination, the first stage of the mating reaction, were generally used. Pretreatment of mt⁺ gametes with the membrane permeable –SH reducing agent dithiothreitol (DTT) inhibits flagellar sexual signaling at concentrations that do not inhibit any part of the mating reaction of mt^? gametes. Wt mt^? is more sensitive than wt mt⁺ to inhibition by low concentrations of p-chloromercuribenzoate sulfonate (pCMBS), an organic mercurial. The membrane-impermeable reducing agent, reduced glutathione (GSH), also preferentially inhibits wt mt^?. Gam-11 mt^?, a fusion-defective mutant, which has been used to study the sensitivity of the adhesion of the plasma membrane-associated mating structures, is less sensitive to GSH and pCMBS inhibition that is wt mt^?. DDT and pCMBS cause an increase in mating structure adhesion in pretreated gam-11. The differential inhibition of pair and group formation during gam-11 × wt mt⁺ matings has suggested a possible mechanism for mating structure adhesion.     

Biochemical and physiological properties of a protein inducing protoplast release during conjugation in theClosterium peracerosum-strigosum-littorale complex

When mating-type minus (mt⁻) and plus (mt⁺) cells of theClosterium peracerosum-strigosum-littorale complex were mixed together in a nitrogen-deficient mating medium, cells of both types released protoplasts, this release being the first step in the process of conjugation. Release of protoplasts by mt⁻ cells also proceeded without pairing in a medium in which mt⁻ and mt⁺ cells had previously been cultured together. A protein with the ability to induce the release of protoplasts was purified from this medium by sequential column-chromatographic steps, and named PR-IP (protoplast-release-inducing protein). The PR-IP had an apparent molecular mass (M_r) of 95000 on gel filtration and could be separated into several isoforms by anion-exchange chromatography. Each isoform consisted of two glycopolypeptides of M_rs 42000 and 19000, while the deglycosylated polypeptides had M_rs of 34000 and 18000, respectively. From an analysis of dose-response curves, the numbers of PR-IP molecules required for the release of a protoplast by a single cell was calculated as 1.5·10⁹ and the concentration required for 50% of the maximum response (ED₅₀) as 4.1·10⁻⁹M. We suggest that the PR-IP is a biologically active glycoprotein which induces the release of gametic protoplasts from mt⁻ cells of thisClosterium complex.     

Mating receptor complex in the flagellar membrane of Chlamydomonas eugametos gametes

Summary The protein composition of the flagellar membrane of C. eugametos mt ^–gametes was analyzed using SDS-polyacrylamide gel electrophoresis. The association of the proteins with the membrane was assessed by differential extraction and an assay for glycosylation. Particular attention was paid to integral membrane proteins that could be associated with the mt ^– agglutinin, the membrane-bound sexual receptor by which the mt ^– gamete binds to its mt ⁺ partner. This agglutinin is a peripheral membrane glycoprotein and must be bound to the flagellar surface by an integral membrane anchor protein that connects the agglutinin with the cell's interior. Immunoaffinity chromatography was performed using Mab 66.3, a monoclonal antibody specific for the mt ^– agglutinin, in order to isolate protein complexes consisting of agglutinin molecules and associated components. Only one integral membrane glycoprotein (M_r = 125 kDa) was isolated that has an association with the agglutinin. It did not bind Mab 66.3, but did bind the lectin wheat germ agglutinin. This was an expected property of the membrane anchor protein because previous research (Kooijman et al. 1989) has shown that cross-linking a WGA-binding glycoprotein by this lectin induces sexual responses that are similar to those induced by agglutinin-agglutinin interactions during mating. We conclude that the 125-kDa glycoprotein is the membrane anchor for the agglutinin.Abbreviations BSA Bovine serum albumin - CBB Coomassie Brilliant Blue - CHAPS 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate - GTC guanidine thiocyanate - mt ^–/mt ⁺ mating type minus/plus - PAS periodic acid Schiff - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TBS TRIS-buffered saline - WGA wheat germ agglutinin     

Sexual agglutinin of mating-type minus gametes in Chlamydomonas reinhardii : I. Loss and recovery of agglutinability of gametes treated with EDTA

The effect of EDTA on the mating-type-specific agglutinins located on the flagellar surfaces of Chlamydomonas reinhardii gametes was investigated. The mating-type minus (mt-) gametes lost their agglutinability without apparent loss of motility soon after addition of EDTA at low concentrations (1-2 mM). At the same time, the cells released into the medium agglutinins which can elicit agglutinative responses of mating-type plus (mt+) gametes specifically. When EDTA was neutralized with Mg2+ or removed by centrifugation, the mt- cells quickly replaced agglutinins by protein synthesis: the recovery process was sensitive to cycloheximide, but not to tunicamycin. The EDTA-treated mt+ gametes lost their agglutinins much more slowly than the mt- gametes. The replacement of mt+ agglutinins was inhibited by both cycloheximide and tunicamycin.