Biophysical characterization of a transit peptide directing chloroplast protein import.
We have investigated the biophysical properties of a 35 amino acid peptide representing the entire length of a chloroplastic targeting sequence. The peptide, termed gamma-tp, corresponds in sequence to the transit peptide of the gamma subunit of the chloroplast ATP synthase from Chlamydomonas reinhardtii. We found that gamma-tp blocks the import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase into isolated pea chloroplasts (KI approximately 5 microM), suggesting that it interacts with higher plant plastids in a physiological manner. We also found the gamma-tp to have a high affinity for nonpolar environments, but not to cause a general disruption of membrane integrity. Hydrophobic moment analysis suggests that the gamma-tp can adopt an amphipathic beta structure. However, circular dichroism measurements indicate that the peptide is largely a random coil, in both the presence and absence of sodium laurylsulfate micelles. In the absence of a recognizable secondary structural targeting motif, we asked whether the presence of a transit peptide on a chloroplast protein increases the protein's overall affinity for nonpolar environments. Phase-partition experiments with Triton X-114 suggest that this is not the case. These results are discussed in relation to the mechanism of protein targeting to chloroplasts.
Theg
S M
SM
Department of Botany, University of California, Davis 95616.
Geske
F J
FJ
D020418:Algal Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D055592:Biophysical Phenomena; D001703:Biophysics; D002463:Cell Membrane Permeability; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000201:enzymology / Q000378:metabolism*; D002942:Circular Dichroism; D003902:Detergents; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D017830:Octoxynol; D011092:Polyethylene Glycols; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism
D020418:Algal Proteins; D060365:Chloroplast Proteins; D003902:Detergents; D021382:Protein Sorting Signals; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D006180:Proton-Translocating ATPases; D012273:Ribulose-Bisphosphate Carboxylase