Deletion of the carboxyl-terminal portion of the transit peptide affects processing but not import or assembly of the small subunit of ribulose-1,5-bisphosphate carboxylase.
Import of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase into the chloroplast has been proposed to involve two proteolytic cleavages which convert the 20-kDa precursor (pSSU) into the mature 14-kDa subunit (SSU) via an 18-kDa intermediate. A deletion mutant (PSd48/57) of pSSU which lacks 10 amino acids in a conserved region in the carboxyl-terminal portion of the transit peptide is converted into a series of 16-18-kDa polypeptides in addition to the mature 14-kDa SSU when imported into isolated pea chloroplasts. We examined import and processing of this mutant pSSU to determine whether the 16-18-kDa SSUs undergo further maturation in the chloroplast stroma to yield 14-kDa SSU. The ratio of incorrectly processed to 14-kDa SSU is stable up to 60 min following import. This indicates that processing of PSd48/57 involves a single proteolytic cleavage which occurs during or immediately following transit across the chloroplast envelope. The carboxyl-terminal portion of the transit peptide confers either sequence specificity for the processing protease or provides a three-dimensional structure necessary for consistent cleavage at the mature amino terminus of SSU. Incorrectly processed SSUs were incorporated into the holoenzyme demonstrating that removal of the entire transit sequence is not necessary for assembly of the holoenzyme.
Ostrem
J A
JA
Department of Biochemistry, University of Arizona, Tucson 85721.
Ramage
R T
RT
Bohnert
H J
HJ
Wasmann
C C
CC
D000595:Amino Acid Sequence; D001692:Biological Transport; D002736:Chloroplasts / Q000201:enzymology*; D004252:DNA Mutational Analysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D012273:Ribulose-Bisphosphate Carboxylase