In vivo regulatory phosphorylation of novel phosphoenolpyruvate carboxylase isoforms in endosperm of developing castor oil seeds.
Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) from developing castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterial PEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conserved p107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated in COS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purified PEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion. Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 by decreasing its K(m)(PEP) and sensitivity to L-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107 phosphorylation increased PEPC2's K(m)(PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylation of p107 was promoted during COS development (coincident with a >5-fold increase in the I(50) [malate] value for total PEPC activity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylated in planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylation status of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.
Tripodi
Karina E
KE
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Turner
William L
WL
Gennidakis
Sam
S
Plaxton
William C
WC
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012278:Ricinus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012639:Seeds / Q000201:enzymology / Q000254:growth & development
D007527:Isoenzymes; D010730:Phosphoenolpyruvate Carboxylase