The timely breakdown of the extracellular matrix by proteolytic enzymes is essential for development, morphogenesis and cell proliferation in plant and animal cells. Sporangin of the unicellular green alga Chlamydomonas reinhardtii that mediates breakdown of the sporangial cell wall to liberate the daughter cells after cell division is characterized as a subtilase-like serine protease. The sporangin gene is specifi cally transcribed during S/M phase in a synchronized vegetative cell cycle. In immunoblot analyses using a polyclonal antibody raised against the sporangin polypeptide, the enzyme is synthesized after mitotic cell division and accumulated in the daughter cells before hatching. Immunofl uorescence analyses showed that sporangin is localized to the fl agella of the daughter cells within the sporangial cell wall, and released into the culture medium. The data suggest that sporangin is released from fl agella concurrently with the digestion of sporangial cell wall, and then the daughter cells are hatched from the sporangia in the Chlamydomonas vegetative cell cycle. Abbreviations: ECM , extracellular matrix ; EST , expressed sequence tag ; GST , glutathione S -transferase ; PBS , phosphatebuffered saline ; RACE , rapid amplifi cation of cDNA ends .The nucleotide sequence of sporangin cDNA has been deposited in the EMBL/Genbank database under the accession number AB303361.

Abstract. Adhesion between Chlamydomonas reinhardtii gametes generates a rapid rise in cAMP levels which stimulates mating responses and zygotic cell fusion (Pasquale and Goodenough, 1987). We show here that sexual adhesion in vivo results in a twofold stimulation of flagellar adenylyl cyclase activity when the enzyme is subsequently assayed in vitro, a stimulation that is specifically blocked by Cd 2÷. A twofold stimulation is also elicited by the in vitro presentation of soluble cross-linking reagents (antisera and concanavalin A). In contrast, the 10-30-fold stimulation of the flagellar cyclase by in vitro exposure to 40°C, first described by Zhang et al. (1991), is insensitive to Cd 2÷ but sensitive to such drugs as trifluoperizine and dibucaine. The capacity for twofold stimulation is displayed by the vegetative and gametic enzymes but is lost when gametes fuse to form zygotes; in contrast, the 10-fold stimulation is displayed by the gametic and zygotic enzymes but not the vegetative enzyme. The signal-defective mutant imp-3 fails to generate the normal mating-triggered cAMP production and can be rescued by exogenous dibutyryl cAMP. It displays normal basal rates of flagellar cyclase activity and a normal twofold stimulation by sexual adhesion and by soluble cross-linkers, but it is defective in 40°C activation. The gametic cell-body adenylyl cyclase is stimulated when wild-type fagella, but not imp-3 flagella, undergo adhesive interactions in vivo, and it can be directly stimulated in vitro by cAMP presentation. We propose that the two levels of fagellar cyclase stimulation reflect either sequential steps in the activation of a single cyclase enzyme, with imp-3 blocked in the second step, or else the sequential activation of two different fagellar enzymes, with imp-3 defective in the second enzyme. We further propose that the cell-body enzyme is activated by the cAMP that is generated when flagellar cyclase activity is fully stimulated.

Cells of Chlamydomonas reinhardtii undergo gametogenesis to produce sexually competent gametes under nitrogen-starved conditions. By using a synchronized system for gametogenesis of early G1 cells, several previously identified marker genes and 18 novel nitrogen-starved gametogenesis (NSG) genes isolated by macroarray analysis were placed into at least three temporal classes of expression. Early genes are induced transiently in the first 2 h after transfer to nitrogen-free medium. Middle genes are strongly induced between 3 h and 4 h after nitrogen removal, a time corresponding to the acquisition of mating competency, suggesting their involvement in the gamete program. Late genes are induced between 5 h and 8 h after nitrogen removal, a time after the completion of gametic differentiation, suggesting that they are not directly involved in the formation of sexually competent gametes. All of the 18 NSG genes examined are induced in both mating-type plus and minus gametes and about two-thirds of the genes are also expressed in the mitotic cell cycle, especially at S/M phases.

A gamete lytic enzyme (GLE) of Chlamydomonas reinhardi is a zinc metalloprotease and mediates digestion of the cell walls of the two mating-type gametes during mating as a necessary prelude to cell fusion. The nucleotide sequence analysis of a cDNA revealed that GLE is synthesized in a preproenzyme form, a 638-amino acid polypeptide (Mr, 69,824) with a 28-amino acid signal peptide, a 155-amino acid propolypeptide, and a 455-amino acid mature polypeptide (Mr, 49,633). A potential site for autocatalytic activation was contained within the propolypeptide and a zinc binding site found within the mature polypeptide; both sites were highly homologous to those in mammalian collagenase. A putative calcium binding site was present in the near C-terminal region of the mature GLE. Both propolypeptide and mature polypeptide had potential sites for asparagine-linked glycosylation, and the Arg-(Pro)3 and Arg-(Pro)2 motifs, which are known to exist in hydroxyproline-rich glycoproteins of the Chlamydomonas cell wall. Northern blot analysis revealed that steady-state levels of the 2.4-kilobase GLE mRNA increased during growth and mitotic cell division in the vegetative cell cycle and also increased markedly during gametogenesis under nitrogenstarved conditions.The controlled remodeling and breakdown of the cell's extracellular matrix (ECM) are important in such biological processes as growth, development, fertilization, and cell fusion in both animals and plants.

No abstract