The Effects of Cyclodextrin Mediated Lipid Depletion of the Chloroplast Outer Envelope on the Import of the Small Subunit of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase

Chloroplasts have a genome that encodes a minority of proteins necessary for organelle function. The rest are encoded in the plant cell nucleus, translated in the cytosol, and post-translationally imported into the chloroplast. Nuclear encoded chloroplast precursor proteins are made as a precursor which contains an amino-terminal extension known as the transit peptide. Transit peptides are believed to be necessary and sufficient for proper targeting. Several studies have suggested that the initial chloroplast binding interaction is between the transit peptide and the lipids of the chloroplast outer chloroplast envelope. In this study, cyclodextrins are utilized to examine the role of the outer membrane lipid constituency on import of the nuclear encoded precursor, prSSU.

Cyclodextrin mediated lipid depletion affects binding and import of prSSU. Cyclodextrins cause only slight lysis, and treated plastids appear normal by electron microscopy and their ability to evolve oxygen. CD pretreatment enhances the ATP-dependent binding of prSSU, yet has no effect on non-specific binding. Cyclodextrins attenuated in vitro import of prSSU into both pea and spinach chloroplasts in a dose dependent fashion. In addition, in vitro protein import decreased as the time length of CD pretreatment increases. As the temperature of pretreatment increases from 0 to 20°C, the effect of cyclodextrin alpha lessens, but cyclodextrin beta enhances import inhibition. Treatment with cyclodextrins did not alter the proteinaceous character of the outer membrane. Electron micrographs show no change in chloroplast ultrastructure with CD treatment and photosynthetic activity is not diminished. Density gradient centrifugation of purified envelopes shows an increase in the density of mixed envelopes after CD treatment indicating a decrease in the lipid to protein ratio. TLC shows that cyclodextrin alpha preferentially removes polar lipids, but not MGDG while beta removes galactolipids, SQDG, and PC but not PG from the outer membrane at the conditions tested. By treating the chloroplasts with cyclodextrins, we have changed the properties of the chloroplast envelopes in such a way that compromises the protein translocation process. Whether this inhibition is a result of a change in lipid to protein ratio, fluidity of the membrane, or bilayer to non-bilayer forming lipid ratio will require further study.