Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 310-313, January 1975 Cell-Cell Interactions: Enhancement of Glycosyl Transferase Ectoenzyme Systems During Chlamydomonas Gametic Contact (cell surfaces/cell recognition and adhesion/enzyme-substrate complexes/aggregation) ROBERT J. McLEAN* AND H. BRUCE BOSMANNt * Department of Biological Sciences, State University College, Brockport, New York 14420; and t Department of Pharmacology and Toxicology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 Communicated by Harold C. Bold November &, 1974 ABSTRACT Glycosyl transferase ectoenzyme systems flagella (9). This meant that the recognition substance was that transfer galactose, glucose, N-acetylglucqsaipine, N- small in comparison to the 108-dalton particles described by Mcetylneuraminic acid, mann6se, and fucose have been de- tected on vegetative cells and gametes of Chilamydomona4 Forster et al. (8). From this observation we tested the hy- moewusii. Gametes have higher levels of activity of the pothesis of whether glycosyl transferases and acceptors were transferase ectoenzyme systems than morphologically present on the external cell surface of the gamete, since such identical vegetative cells, as determined by transfer of ectoenzyme systems have been postulated as a potential monbsaccharide onto endogenous cell surface acceptors. cell recognition and adhesion (5-7). When (+)-and (-) gametes kire mixed, there is a significant mechanism mediating increase in the activity of transfekase ectoenzymS systems. Our data demonstrate that glycosyl transferase and ac- No enhancement in activity of transferase ectoenzyme ceptors are present on the external surfaces of the (+) and systems occurs when (+) and (7) vegetatie' cells are (-) mating types of Chlamydomonas moewusii gametes, and mixed. Flagellar membrane yesieles obtained from (+) that when flagellar membrane vesicles or gametes of the two and (-) gametes show high activity of transferase ecto- enzyme- systems per mg of protein and also demonstrate mating types are mixed, there is a significant increase in the enhanced activity upon mixing. Therefqre, glycosyl trans- activity of the glycosyl transferase ectoenzyme system$. ferases and acceptors seem to be located on ihe flagellar It would appear that external surface glycosyl transferases membrane and appear to have a function particularly re- and appropriate specific cell surface acceptors are directly im- lated to gametic cells. The mechanism of cellular adhesion plicated in a function related to cellular recognition and/or or recogkiition proposed by Roseman (1970, Chem. Phys. Lipids 5I 270-297), involving glycogyl transferases and ac- adhesion. ceptors, is strongly suggested by our data for the mating reaction in Chlamydomonas. MATERIALS AND METHODS The (+) and (-) mating types of the unicellular flagellate, Glycosyl transferase and acceptor molecules have been de- Chlamydomona2 moewusii, nos. 96 and 97, respectively (10), tected on the external cell surface of several different mam- were grown on growth medium agar (11) for 8-10 days at malian cells (1-6), but their function at this external surface 250 i 10 in north light. Cells were then suspended in liquid is uncertain. Roseman (7) has postulated that these ecto- induction medium (11) to induce gametogenesis. In order to enzyme systems may be responsible for cell recognition and/or obtain motile vegetative cells (cells not responsive in the mat- adhesiveness by the formation of an enzyme-substrate com- ing reaction), liquid growth medium was supplemented with plex. Such a specific complex formation could provide the 0.1% (w/v) NH4C1 (12). All cultures were axenic, and aseptic high degree of specificity required in such phenomena as techniques were used during induction. histotypic compatibility and egg-sperm recognition. Addi- Vegetative cells are not distinguishable from gametes ex- tionally, completion of the reaction could break the enzyme- cept that gametes of one mating type will adhere to gametes substrate complex, thus explaining the modulation between of the opposite mating type; i.e., (+) gametes will adhere to adhesion and disadhesion found in many cell interactions, e.g., (-) gametes, but (+) gametes will not adhere to (-) vegeta- release between mammalian cells observed during contact in- tive cells. Additionally, (+) and (-) vegetative cells have no hibition. affinity for each other (12). Therefore, vegetative cells were We have investigated a protist system, that of the Chlamy- used as controls in some experiments for comparison with domonas mating reaction, in which flagella of opposite gametes gametes. adhere to each other by their flagellar membrane surfaces. Membrane vesicles were isolated by differential centrifuga- After a cytoplasmic bridge has formed between the cell tion from the induction medium (9) after gametes had been bodies of two opposite gametes, the adhering flagella are re- incubated for 24-28 hr. Suspended membrane material was leased from each other and are no longer agglutinable with passed through a 1.0-jum filter (Nuclepore Corp., Pleasanton, other gametes. The mechanism of gametic adhesion and Calif.) and then layered over 40% sucrose and centrifuged for recognition in Chlamydomonas is not known but was ascribed 1 hr at 16,300 X g. Membranes were collected at the interface to large glycoprotein particles attached to the flagellar sur- and washed twice. face that were easily lost in the cell medium. Particles from the cell medium of one mating type could cause isoagglutina- t Glycosyl transferase ectoenzyme system is the term used tion (agglutination within one mating type) when added to herein to denote external surface glycosyl transferases and opposite gametes (8). It was recently found that the active surface acceptors whose activity is measured as a system. That is, factor in the cell medium was actually membrane vesicles, under the conditions of assay either glycosyl transferase or ac- and no large, active particles were observed on the vesicles or ceptor could be limiting kinetically in the reaction. 310 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Cell Recognition and Glycosyl Ectoenzymes 311 Assay procedures for glycosyl transferase and particulars TABLE 1. Activity of the surface and total glycosyl transferase concerning substrates (preparation, specific activity, etc.) ectoenzyme system of isolated active vesicles of followed those reported (1, 2, 13). Gametes and vesicles were Chlamydomonas gametes washed in induction medium, and vegetative cells were washed in growth medium. The assay mixture for surface activity 0.5 Volume contained 10 MAl of the nucleotide phosphate [14C]mono- strain 96 plus 0.5 saccharide (about 10-9 mol), 10 of 0.1 M MgCl2, 10 of Al 1M Strain Strain 0.1 M 10 of 0.1 M Tris HCl and 50 volume MnCl2, MuI * (pH 7.6), Mul of Substrate 96(+) 97(-) strain 97 the active vesicles (about 10lg of protein) or cells suspended in 0.145 M NaCl. The final pH of the reaction was 6.9. After Surface activity 30 min of incubation at either 250 or 37°, 3 volumes of 1% UDP-galactose 3,810 4,409 16,888 phosphotungstic acid in 0.5 M HCl were added and the UDP-glucose 5,211 4,687 19,460 precipitate was centrifuged out of solution. The precipitate UDP-N-acetylglu- was washed twice with 10% trichloroacetic acid and once cosamine 5,612 5,024 21,872 with ethanol-diethyl ether (2:1, v/v), and the resultant CMP-N-acetylneur- aminic acid 6,800 6,100 49,072 precipitate was dissolved in 1 M NaOH. was Radioactivity GDP-mannose 5,321 5,916 29,112 determined in a liquid scintillation counter. Data are ex- GDP-fucose 3,507 4,111 19,100 pressed as cpm per mg of protein with background sub- tracted. Protein was determined by the method of Lowry et al. Total activity (14). For assay of the total glycosyl transferase system, a UDP-galactose 4,968 5,609 6,311 0.1% Triton X-100 homogenate of cells or vesicles (sonicated, UDP-glucose 6,409 6,802 7,119 UDP-N-acetylglu- then given 30 strokes in a Ten Broeck homogenizer) replaced cosamine 7,219 6,084 10,897 whole cells or suspended vesicles in the assay. This procedure CMP-N-acetylneur- allowed for a total assay of both internal and surface glycosyl aminic acid 12,462 10,492 16,471 transferase systems. GDP-mannose 7,996 8,142 12,489 Measurements of electrophoretic mobilities were done ac- GDP-fucose 4,079 4,822 5,967 cording to procedures outlined (2), with a whole-cell particle electrophoresis chamber obtained from Rank Brothers, Vesicles were prepared as described in the text. Data are cpm/ Bottisham, U.K. Mobilities of the particles were calculated mg of protein. The surface activity system contained 10 Ml of the in (,Mm/sec) V- cm-'. At least 160 individual cells were nucleotide phosphate [14C]monosaccharide (about 10-9 mol), 10 analyzed for each cell type for the electrophoretic mobility MAl of 0.1 M MgCl2, lOAl of 0.1 M MnCl2, 1O Il of 0.1 M Tris.HCl measurements. (pH 7.6), and 50 Ml of the active vesicles (about 10 g of protein) suspended in 0.145 M NaCl. The final pH of the reaction was 6.9. After 30 min of incubation at (reaction at RESULTS 370 250 gave quanti- tatively similar results), macromolecular-bound radioactivity was Isolated flagellar membrane vesicles from gametes were as- determined. For the total system, 50 MAl (about 10 ug of protein) sayed for activity of the glycosyl transferase ectoenzyme sys- of a 0.1% Triton X-100 homogenate (sonicated, then given 30 tem. Table 1 shows activity of surface glycosyl transferase strokes in a Ten Broeck homogenizer) replaced the suspended ectoenzyme of vesicles and total transferase activity of vesicles vesicles in the assay. Since only small amounts of protein were available, each assay resulted in solubilized with 0.1% Triton X-100. Total activity of in- cpm that ranged from 3 to 50 dividual transferases is usually less than double that of the times background (19 cpm).