Biochemical Characterization of Wild-Type and Mutant Isoamylases of Chlamydomonas reinhardtii Supports a Function of the Multimeric Enzyme Organization in Amylopectin Maturation

Abstract

(A.M.M.) Chlamydomonas reinhardtii mutants of the STA8 gene produce reduced amounts of high amylose starch and phytoglycogen. In contrast to the previously described phytoglycogen-producing mutants of C. reinhardtii that contain no residual isoamylase activity, the sta8 mutants still contained 35% of the normal amount of enzyme activity. We have purified this residual isoamylase and 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 one-half. This coincides with the disappearance of two out of the three activity bands that can be seen on zymogram gels. Wild-type and mutant enzymes are shown to be located within the plastid. In addition, they both act by cleaving off the outer branches of polysaccharides with no consistent difference in enzyme specificity. Because the mutant enzyme was demonstrated to digest phytoglycogen to completion in vitro, we propose that its inability to do so in vivo supports a function of the enzyme complex architecture in the processing of pre-amylopectin chains.

Crystal Structure of the Chlamydomonas Starch Debranching Enzyme Isoamylase ISA1 Reveals Insights into the Mechanism of Branch Trimming and Complex Assembly

“…We subsequently wanted to confirm the nature of the larger oligomeric assemblies in which ISA1 participates in vivo (17). As the Chlamydomonas sta8 mutants display similar starch characteristic to the isa2 mutants of certain plant species, we wanted to clarify the relationship of the STA8 locus to the ISA2 protein.…”

“…Under nitrogen starvation conditions, Chlamydomonas strains carrying mutations at either the STA7 and STA8 loci display phenotypes similar to that of the respective ISA1 and ISA2 mutants observed in the cereal species (7,17). The STA7 locus was proven to define the structural gene of the algal ISA1 subunit.…”

“…A full account of starch characterization procedures including amyloglucosidase assays, starch purification on Percoll gradients, (17), respectively. In each case, 100 g of total protein extract were loaded on the gels.…”

“…Mutation of the STA7 locus leads to a very severe reduction of starch content and its replacement by a water-soluble polysaccharide phytoglycogen (7). Although not as severe as the sta7 mutants, mutants of the STA8 locus have also been described to accumulate both phytoglycogen and a reduced amount of high amylose starch (7,17). In nitrogen-supplied medium, however, whereas the sta7 mutants remain starchless (18), the sta8 mutants display no reduction in starch content and phytoglycogen accumulation is strongly reduced (19).…”

Functional Interactions between Starch Synthase III and Isoamylase-Type Starch-Debranching Enzyme in Maize Endosperm

“…These encode ISA1 and ISA2, which are components of a heteromeric complex. Both proteins are necessary for enzymatic function, explaining why mutation of either gene causes the same phenotype (Dauvillée et al, 2001a(Dauvillée et al, , 2001bHussain et al, 2003;Bustos et al, 2004;Delatte et al, 2005;Delvallé et al, 2005). As in other species, ISA1 is necessary for normal starch biosynthesis in cereal endosperm (James et al, 1995;Kubo et al, 1999;Burton et al, 2002); however, the lack of ISA2 does not cause phytoglycogen accumulation (Kubo et al, 2010;Utsumi et al, 2011).…”