Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ.

Abstract

Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the aggutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reductionJalkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination.The first cell-cell recognition mechanisms developed by eukaryotes were probably used in fertilization. It follows that the modern sexual protozoa, although clearly "highly evolved" in their own right, may offer clues as to what kinds of cell surface molecules were originally used to mediate gametic fusion with the appropriate partner. In the unicellular alga Chlamydomonas reinhardi, the key sexual recognition molecule has been shown to be a large hydroxyproline-rich fibrous glycoprotein (1, 2). Since similar proteins appear to function in the organization of the extracellular matrix in higher plants (3), and since the collagens, also rich in hydroxylated amino acids, are widely implicated in organizing the tissues of the metazoa (4), an understanding of the Chlamydomonas agglutinin structure may yield evolutionary insights into the origins of cell-cell interactions in eukaryotes.This paper first presents a detailed analysis of the purified agglutinin protein using the quick-freeze, deep-etch techniques developed by Heuser (5,6). We compare the structure 924 niques developed by Heuser (5, 6). We compare the structure of the mating-type plus (rot +) and mating-type minus (rot-) proteins, and describe alterations in their structure induced by proteolytic digestion and disulfide reduction/alkylation. In the course of this study we h...

Characterization of the purified Chlamydomonas minus agglutinin.

“…4, lanes 1 and 4). As described in detail elsewhere (15), one or more faster-migrating glycopolypeptides, collectively designated B (Fig. 4, lanes 2-4), often contaminate peak agglutinin fractions; since the B material alone has no adhesive activity, however, it is not relevant to the present study.…”

“…2 (lanes 2 and 3) demonstrates that both mutants lack a polypeptide A-component, which supports the identification of this high molecular weight glycoprotein as the active minus agglutinin molecule. In addition, molecules with the morphology of A-are present on the surface of gametic, but not vegetative, minus cells when observed in situ (15).…”

“…2). These species, which are also produced by wild-type gametes, derive from the flagellum but are not involved in adhesion; they are described in detail elsewhere (15). EDTA extracts prepared from wild-type rot-and agglutination-defective mutant (imp-10 and imp-12) "gametes" (3 x 1011 cells per sample) were fractionated by two chromatographic runs over Fractogel-75 (as in Fig.…”

“…(4). Quick-frozen, deep-etched samples of purified agglutinins were prepared for electron microscopy according to Heuser (14) and visualized as described previously (5,7,15).…”

Mutations Affecting O-Glycosylation in Chlamydomonas reinhardtii Cause Delayed Cell Wall Degradation and Sex-Limited Sterility

“…is responsible for the selectivity of the sexual recognition and fusion processes). The mechanism for this selectivity rests in part on the presence at the surface of the gametic flagellae of mating type-specific agglutinins (reviewed by Adair, 1985). Agglutinins are high M , glycoproteins with strong similarities to cell wall glycoproteins in their shape, amino acid composition, and glycosylation pattern (Cooper et al, 1983).…”