Gametes of the unicellular green alga Chlamydomonas eugametos agglutinate via their flagella. The mating type plus agglutination factor was solubilized by relatively mild treatments such as a short pH shock or an osmotic shock indicating that it is an extrinsic 'membrane component. It was also extracted in the nonionic detergent Triton X-100. A simple two-step procedure consisting of gel filtration over Sepharose 4B-cross-linked followed by anion exchange chromatography of the void volume yielded an electrophoretically pure preparation of a single high molecular weight glycoprotein. The agglutination factor sedimented as a 93 S particle (assuming a density of 1.50) in sucrose gradients. This low value, compared with the high apparent molecular weight seen during gel filtration and electrophoresis, suggests that the agglutination factor is a rod-like molecule. This was confirmed by viewing rotary-shadowed preparations in the electron microscope. A population of long slender molecules was revealed (328 ± 20 nanometers), many of which had a knob at one end and a flexible region about one fourth of the length from the other end.Cell-cell recognition in plants plays a role in a remarkable range of phenomena including sexual reproduction, parasitism, grafting, and many others (15). The sexual agglutination process shown by gametes of Chlamydomonas represents a spectacular example of such a recognition process (11,27,28 . More recently, the isolation of the mtV agglutination factor was also reported (6, 24). In C. eugametos, a species that cannot sexually agglutinate with C. reinhardtii, the mtr agglutination factor has been purified and analyzed by Musgrave et al. (19) and by Homan (12). In this paper we report the purification of the mt+ agglutination factor ofthis species, and show that it is a long linearglycoprotein.
MATERIALS AND METHODSCell Cultures. Chlamydomonas eugametos strains 11-5/9 (mt+) and 11-5/10 (mtr) from the Sammlung von Algenkulturen, Gottingen, Federal Republic of Germany, were cultivated in Petri dishes on agar-containing medium in a 12-h light/12-h dark regimen as described by Mesland (17). Gamete suspensions were obtained by flooding 2-to 3-week-old cultures with distilled H20 just before the start of the dark period. They were harvested the following light period. A Petri dish culture contained on the average 2.5 x 108 cells. Suspensions of vegetative cells were obtained by flooding with 0.5% (w/v) NH4Cl (23).Detergent Extraction of mt' Agglutination Factor. Gamete suspensions were centrifuged at 5°C at 1500g for 15 min. The cells were resuspended in ice-cold 0.1I% (w/v) Triton X-100 (1 ml/culture) and gently shaken at 5°C for 1 h. The suspension was then centrifuged at 48,000g for 30 min. The supernatant was collected and mixed with extensively washed Amberlite XAD-2 (20 g wet weight/100 ml) to remove Triton X-100 (4). The Amberlite suspension was shaken at 5°C for 15 min and filtered to remove the Amberlite. Fresh Amberlite was added to the filtrate and the procedure was repeated. T...