Organelle-Bound Malate Dehydrogenase Isoenzymes Are Synthesized as Higher Molecular Weight Precursors

Abstract

Biosynthesis of malate dehydrogenase isoenzymes was studied in cotyledons of watermelons (Citrudlus vulgaris Schrad., var. Stone Mountain). The glyoxysomal and mitochondrial isoenzymes are synthesized as higher molecular weight precursors which can be immunoprecipitated by monospecific antibodies from the products of in vitro translation in reticulocyte lysates programed with cotyledonary mRNA and with the same size from enzyme extracts of pulse-labeled cotyledons. During translocation from the cytosol into the organelles, processing takes place. An 8 kilodalton extra sequence is cleaved from the glyoxysomal precursor and a 3.3 kilodalton extra sequence from the mitochondrial precursor producing the native subunits of 33 and 38 kilodaltons, respectively. The data support a posttranslational translocation of the organelle-destined malate dehydrogenase isoenzymes. The in vitro translation of the cytosolic malate dehydrogenase I yields a product which has the same molecular weight as the subunit of the native isoenzyme (39.5 kilodaltons).distinguished by electrophoretic or serological techniques. Earlier studies on the in vitro synthesis of gMDH by a wheat germ system by our group (26) had shown that this isoenzyme is made as a higher mol wt polypeptide compared with the subunit of gMDH isolated from watermelon cotyledons thereby suggesting the need for processing during the importation into the glyoxysomes. The results were confirmed subsequently by Riezman et al. (20) for cucumber cotyledons.We now present a comparative study on the in vitro synthesis of gMDH, mMDH, and cMDH I using the heterologous reticulocyte system. Our results indicate that mMDH also is synthesized as a larger in vitro product3 in contrast to the cytosolic isoenzyme.Pulse labeling experiments provided evidence that the higher mol wt MDH polypeptide precursors were also detectable under in vivo conditions. The posttranslational implications for the role of such precursors in directing transport into glyoxysomes and mitochondria are discussed. MATERIALS AND METHODSThe development of cell organelles requires the import of proteins which are synthesized on cytoplasmic ribosomes. The translocation from the site of synthesis to the site of function involves a highly specific recognition mechanism by the organelle envelope which only accepts the organelle-destined proteins for segregation within the organelle.The use of isoenzyme system for translocation studies requires that individual isoenzymes be located in different organelles within the same cell. Differences in these proteins or their precursors should be related, at least in part, to the pattern of distribution. Only a very few isoenzyme systems have been employed in this way, e.g. the aspartate aminotransferase (17) and the malate dehydrogenase (19) from rat liver where the mitochondrial and the cytosolic isoenzymes were compared. In vitro transport studies with purified isoenzymes revealed that the high selectivity of the isoenzyme sequestration by the mitochondria could be relate...

Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide.

“…3)-to encode gMDH. The deduced molecular mass of the preform of the subunit of watermelon gMDH is 37.6 21) and to 41 kDa upon translation in the reticulocyte cell free protein synthesizing system (22).…”

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

“…Alternative splicing phenomena are frequently related to isozyme production (Milner-White, 1984). On the other hand, mitochondrial MDH has been shown to act as a large precursor which might form different isozymes with differential posttranslational modifications (Gietl and Hock, 1982).…”

Mitochondrial malate dehydrogenase from watermelon: sequence of cDNA clones and primary structure of the higher-plant precursor protein

“…Apparent molecular weights determined by gel electrophoresis gave values of 41000 and 38 000, respectively [6]. Alignment of the amino acid sequence of the watermelon mMDH with porcine mMDH [3] and mMDH from rat [9], mouse [33], yeast [35] as well as the MDH from E. coli [20,22] is presented in Fig.…”