Biochemical characterization of wild-type and mutant isoamylases of Chlamydomonas reinhardtii supports a function of the multimeric enzyme organization in amylopectin maturation

Chlamydomonas reinhardtii mutants of the STA8 gene produce reduced amounts of high amylose starch and phytoglycogen. In contrast to the previously des cribed phytoglycogen-producing mutants of C. reinhardtii that contain no re sidual isoamylase activity, the sta8 mutants still contained 35% of the nor mal amount of enzyme activity. We have purified this residual isoamylase an d compared it with the wild-type C. reinhardtii enzyme. We have found that the high-mass multimeric enzyme has reduced its average mass at least by on e-half. This coincides with the disappearance of two out of the three activ ity bands that can be seen on zymogram gels. Wild-type and mutant enzymes a re shown to be located within the plastid. In addition, they both act by cl eaving off the outer branches of polysaccharides with no consistent differe nce in enzyme specificity. Because the mutant enzyme was demonstrated to di gest phytoglycogen to completion in vitro, we propose that its inability to do so in vivo supports a function of the enzyme complex architecture in th e processing of pre-amylopectin chains.