In Vitro Synthesis and Processing of Tomato Fruit Polygalacturonase 1

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The in vitro processing of tomato fruit polygalacturonase (PG) (poly[1,4-α-d-galacturonide]glucanohydrolase, EC 3.2.1.15) was studied. Complete chemical deglycosylation of a mixture of mature, purified PG 2A and PG 2B isozymes (45 and 46 kilodaltons; respectively) with trifluoromethane sulfonic acid yielded a single polypeptide of 42 kilodaltons. Similarly, N-terminal amino acid sequencing of the PG 2A/2B isozyme mixture yielded a single 21 amino acid N-terminal sequence, suggesting that the two isozymes result from differential post-translational processing of a single polypeptide. Translation of PG mRNA in vitro results in the synthesis of a single polypeptide with an apparent molecular weight of 54 kilodaltons. Nucleotide sequence analysis of a full-length PG cDNA clone indicates that the large size difference between the PG in vitro translation product and the mature isozymes is due to the presence of a 71 amino acid (8.2 kilodaltons) domain at the N-terminus of in vitro translated PG, consisting of a hydrophobic signal sequence followed by a highly charged prosequence. To determine the precise cleavage site of the signal sequence, PG mRNA was translated in vitro in the presence of canine pancreas microsomal membranes. This resulted in the production of two glycosylated PG processing intermediates with apparent molecular weights of 58 and 61 kilodaltons. The PG processing intermediates were shown to be sequestered within the lumen of the microsomal membranes by protease protection and centrifugational analysis. Deglycosylation of the PG processing intermediates with endoglycosidase H yielded a single polypeptide with an apparent molecular weight of 54 kilodaltons. The production of two distinct, glycosylated processing intermediates from the single in vitro translated PG polypeptide suggests a mechanism by which the differential glycosylation observed for the mature PG 2A and PG 2B isozymes may occur. Edman degradation of 3H-labeled 58 and 61 kilodalton PG processing intermediates indicates that the site of signal sequence cleavage is after amino acid 24 (serine). These results suggest that the proteolytic processing of PG occurs in at least two steps, the first being the co-translational removal of the 24 amino acid signal sequence and the second being the presumed post-translational removal of the remaining highly charged 47 amino acid prosequence.

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