Functional traits of lipid particles originating from thylakoid membranes

Abstract

Lipid-protein particles bearing the 55 kD Rubisco large subunit (RLSU) and no detectable corresponding Rubisco small subunit (RSSU) were isolated from the stroma of intact Phaseolus vulgaris chloroplasts by flotation centrifugation. Stromal RLSU-bearing particles appear to originate from thylakoids inasmuch as they can also be generated in vitro by illumination of isolated thylakoids. Their formation in vitro is facilitated by light or ATP and can be inhibited by prior heat-denaturation of the membranes. The RLSU-containing lipid-protein particles range from 0.05 to 0.10um in radius, contain the same fatty acids as thylakoids, but have a 10- to 15-fold higher free to esterified fatty acid ratio than thylakoids. RLSU-bearing lipid-protein particles with no detectable RSSU were also immunopurified from the populations of both stromal lipid-protein particles and those generated in vitro from illuminated thylakoids. Protease-shaving indicated that RLSU is embedded in the lipid-protein particles and that there is also protease-protected RLSU in thylakoids. These observations collectively indicate that RLSU associated with thylakoids is released into the stroma by light-facilitated blebbing of lipid-protein particles. The release of RLSU-containing particles may in turn be coordinated with the assembly of Rubisco holoenzyme inasmuch as chaperonin 60 is also associated with lipid-protein particles isolated from stroma.

Two subpopulations of lipid particles isolated from chloroplasts of Phaseolus vulgaris leaves have been found to contain PAP (plastid-lipid-associated protein). One subpopulation is comprised of plastoglobuli isolated from sonicated thylakoids by flotation centrifugation according to a standard procedure. The second subpopulation is comprised of higher density particles, previously referred to as lipid-protein particles (Ghosh et al., 1994), present in the chloroplast stroma. Since these particles contain PAP but have a higher buoyant density than plastoglobuli, they have been termed plastoglobuli-like particles. Of particular interest is the finding that plastoglobuli and plastoglobuli-like particles also both contain catabolites of the thylakoid protein, cytochrome f. These observations support the view that there are distinguishable populations of plastoglobuli-like particles in chloroplasts. They further suggest that the formation of these particles may allow removal of cytochrome f catabolites from the thylakoid membrane that are destined for degradation as part of normal thylakoid turnover.

A lipase gene is up-regulated in the leaves of Phaseolus vulgaris as a function of age and in response to exogenous ethylene, as indicated by Northern blot analysis using a heterologous cDNA as a probe. A series of degenerate primers were designed based on the amino acid and nucleotide sequences of seven plant lipases, and used to amplify putative lipase clones from 3-week-old primary leaf RNA by RT-PCR. Three partial clones were obtained, all of which corresponded to lipases as determined by comparison to protein databanks. Sequence analysis indicates that one or more of the leaf lipases may correspond to a lipolytic acyl hydrolase, may be regulated by phosphorylation and could be targeted to chloroplasts. Lipolytic acyl hydrolases from Phaseolus leaves capable of degrading galactolipids have been described on a biochemical level, but genes for such enzymes have not been isolated.