Molecular characterization of cell wall degrading proteases in Chlamydomonas reinhardtii

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In Chlamydomonas reinhardtii. the release or shedding of cell walls is observed in at least two different stages of the life cycle. First, zoospores, the products of mitosis in the vegetative cell cycle, hatch by breaking of their enclosing sporangial walls with hatching enzyme (HE). Second, gametes of two mating-types dissolve and shed their walls with lytic enzyme (LE) during mating as a necessary prelude to cell fusion. We have previously purified HE and LE and characterized then as a semine and metalloprotease, respectively. In the present work. we have further characterized these two cell wall degrading proteases with respect to their amino-terminal sequences and their cleavage specificities on peptides. We also found a protease inhibitor of RE, which occurred specifically during mitotic cell divisions. The results obtained are as follows:1. The N-terminal amino acid sequence of LE was EIYAGKPIDLRTIVYINDFS. This sequence was the same for LE which exists in vegetative cells, in gametic plus and minus cells and in mating medium after excretion. By this information of amino acid sequence, we have currently tried and succeeded in isolating cDNA clone of LE.2. By using a variety of synthetic model peptides. we found that LE cleaves the peptides bonds between two consecutive hydrophobic amino acids, whereas HE hydrodlyzes peptides at the carboxyl site of Lys or Arg.3. We were unable to find any HE activities in mature sporangia just before zoospore liberation. Instead, we found a specific protease inhibitor for HE in the cell homogenates. Partially purified inhibitor had a molecular mass of about 55 kDa and formed a revesible complex with HE. This inhibitor occurred only during mitotic cell divisions in the vegetative cell cycle and was accumulated in a space between the dividing cells and mother (sporangial) walls.