Molecular characterization of cell wall degrading proteases in Chlamydomonas reinhardtii

在莱茵衣藻中。在生命周期的至少两个不同阶段观察到细胞壁的释放或脱落。首先，游动孢子是营养细胞周期中有丝分裂的产物，通过孵化酶（HE）打破其封闭的孢子囊壁而孵化。其次，两种交配型的配子在交配过程中用溶解酶（LE）溶解并脱落细胞壁，这是细胞融合的必要前奏。我们先前已经纯化HE和LE，并分别表征为精蛋白酶和金属蛋白酶。在目前的工作中。我们进一步表征了这两种细胞壁降解蛋白酶的氨基末端序列和它们对肽的切割特异性。我们还发现了RE的蛋白酶抑制剂，其特异性地发生在有丝分裂细胞分裂期间。主要研究结果如下：1. LE的N端氨基酸序列为EIYAGKPIDLRTIVYINDFS。LE在营养细胞、配子正、负细胞和排泄后的交配培养基中的存在顺序也是一样的。根据氨基酸序列的信息，我们目前已经尝试并成功地分离了LE的cDNA克隆。通过使用多种合成模型肽。我们发现LE切割两个连续疏水氨基酸之间的肽键，而HE水解赖氨酸或精氨酸的羧基位点处的肽。在游动孢子释放前的成熟孢子囊中，我们未能发现任何HE活性。相反，我们在细胞匀浆中发现了HE的特异性蛋白酶抑制剂。部分纯化的抑制剂的分子量约为55 kDa，并与HE形成可逆的复合物。这种抑制剂只发生在有丝分裂细胞分裂的营养细胞周期，并积累在一个空间之间的分裂细胞和母亲（孢子囊）壁。

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.