Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds

Abstract

Plastid pyruvate kinase (PK,) activity and anti-(castor oil seed [COSI PK, ) immunoglobulin C immunoreactive polypeptides were recovered in the stroma but not from envelope membranes of purified COS leukoplasts that had been subfractionated by sucrose density gradient centrifugation. The PK, was highly purified from isolated leukoplasts using anion-exchange and ADP-agarose chromatographies. Proteolysis of PK, was almost entirely eliminated by including 2,2'-dipyridyl disulfide in purification buffers. The final preparation contained 63.5-kD ( a subunit) and 54-kD ( p subunit) polypeptides that stained for protein and cross-reacted with anti-(COS PK, ) immunoglobulin C with similar intensities. These two polypeptides co-eluted following gel-filtration chromatography and co-migrated during nondenaturing isoelectric focusing-polyacrylamide gel electrophoresis. The enzyme's native M, was estimated t o be 334,000. This PK, thus appears to exist as an a,p,-heterohexamer. Comparison of the respective N-terminal sequences of the a and subunits with the deduced amino acid sequences for severa1 PK, cDNAs indicated that (a) the a and p subunits are encoded by COS genes previously designated as PKpA and PKpC, respectively, and (b) respective transit peptides of 4.8-and 5.5-kD are cleaved from the a and p subunit preproteins following their translocation into the leukoplast.

Quantitative Proteomics of Seed Filling in Castor: Comparison with Soybean and Rapeseed Reveals Differences between Photosynthetic and Nonphotosynthetic Seed Metabolism

“…This step is considered to be a highly regulated glycolytic step as it is activated by pH changes, inhibited by Glu/Asp and ATP, and degraded after phosphorylation and ubiquitination (Podesta and Plaxton, 1991;Tang et al, 2003;Turner et al, 2005). Among the oilseeds compared, PK c isoforms were identified in soybean and castor, while the PK p was identified in rapeseed; however, in earlier studies, PK p activity and concentration was shown to coincide with fatty acid levels in developing castor seed (Plaxton, 1991;Negm et al, 1995). Castor and soybean expression patterns are very different.…”

A Heteromeric Plastidic Pyruvate Kinase Complex Involved in Seed Oil Biosynthesis inArabidopsis

“…The reconstitution of active PK p s from individually purified inactive subunits in this study and the in vivo co-IP results leave little ambiguity as to the heteromeric structure of the Arabidopsis PK p s. Some studies of recombinant PK p polypeptides from developing R. communis endosperm concluded that the PK p -a and PK p -b homologs are distinct enzymes (Blakeley and Dennis, 1993;Blakeley et al, 1995;Wan et al, 1995), while others have determined that the same proteins are actually subunits of a single heteromeric PK p complex (Plaxton et al, 1990;Plaxton, 1991;Negm et al, 1995). The data presented in this article support the conclusion that PK p is a complex composed of two different subunits.…”

“…Arabidopsis, for instance, has 14 annotated PK genes that likely exhibit a large degree of variation with respect to regulation of gene expression and enzyme activity (Arabidopsis Genome Initiative, 2000). PK p has been purified and characterized from castor (Ricinus communis) endosperm and B. napus suspension cell cultures (Plaxton et al, 1990Negm et al, 1995). Both enzymes consist of a-and b-subunits and appear to exist as 3a3b heterohexamers.…”

Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm

“…The endosperm and embryo of castor seeds at stages 5 to 7 (Greenwood and Bewley, 1981) were excised and the leucoplasts isolated according to the method of Negm et al (1995). Protease inhibitors were omitted from the buffers.…”

“…Leucoplasts were isolated from developing castor endosperm as described by Negm et al (1995). This method has been shown to yield highly intact leucoplast preparations, which are devoid of contamination from the cytosol and other subcellular compartments (Negm et al, 1995).…”