Article Figures & Data
Figures
- Fig. 1.
Flow chart illustrating the isolation of: A, floated plastoglobuli and higher-density plastoglobuli from sonicated chloroplasts; and B, lipid-protein particles from the stroma of non-sonicated chloroplasts.
- Fig. 2.
SDS-PAGE and western blots of floated plastoglobuli, higher density plastoglobuli, stromal lipid-protein particles, and thylakoids. A, Silver-stained SDS-PAGE gel. Lane 1, Floated plastoglobuli; lane 2, higher-density plastoglobuli (fraction F1); lane 3, higher-density plastoglobuli (fraction F2); lane 4, higher-density plastoglobuli (fraction F3); lane 5, higher-density plastoglobuli (fraction F4); lane 6, stromal lipid-protein particles; lane 7, thylakoids. Lanes were loaded with equal protein (1.2 μg). Molecular mass markers (kD) are indicated. B, Western blot probed with PAP antibody. Lanes are as in A. The upper arrow indicates the position of a 32-kD protein that cross-reacts with PAP antibody. The lower arrow indicates the position of a 28-kD protein that cross-reacts with PAP antibody. C, Western blot probed with polyclonal antibody raised against SDS-PAGE-purified cytochrome f. Lanes are as in A. The thick arrow indicates the position of mature, full-length cytochrome f. Two lower M rcatabolites are indicated by thin arrows.
- Fig. 3.
Immunodetection of cytochrome f and PAP and quantitation of fatty acid co-associated with stromal lipid-protein particles fractionated on a Sephacryl size-exclusion column. A, Western blot probed with polyclonal antibody raised against SDS-PAGE-purified mature, full-length cytochrome f. Lane 1, Fraction 32; lane 2, fraction 33; lane 3, fraction 34; lane 4, fraction 35; lane 5, fraction 36; lane 6, fraction 37; lane 7, fraction 38. Lanes were loaded with equal volume. The thick arrow indicates the position of full-length cytochrome f. LowerM r catabolites are indicated by thin arrows. B, Western blot from A stripped of antibodies used for detection of cytochrome f and reprobed with PAP antibodies. The arrow indicates the position of the 32-kD PAP. Lanes are as in A. C, Total fatty acid content of pooled column fractions.
- Fig. 4.
Fatty acid composition of total lipid extracts from thylakoids, stromal lipid-protein particles, floated plastoglobuli, and higher-density plastoglobuli. Values are expressed as means ± se. 14:0, Myristic acid; 16:0, palmitic acid; 16:1, palmitoleic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; and 18:3, linolenic acid. F1, F2, F3, and F4 are higher-density plastoglobuli supernatant fractions collected sequentially from beneath the floated plastoglobuli.
- Fig. 5.
Western blots demonstrating that the lowerM r forms of cytochrome fassociated with stromal lipid-protein particles and thylakoids are catabolites of the mature protein. A, Schematic representation showing the localization of cytochrome f in the thylakoid membrane (adapted from Gray, 1992). Numbers refer to amino acid residues and the portions of the protein against which the terminus-specific antibodies were raised are indicated by lighter shading. B, Western blot probed with antibody raised against SDS-PAGE-purified full-length cytochromef. Lane 1, Stromal lipid-protein particles; lane 2, thylakoids. The thick arrow indicates the position of mature cytochromef. Thin arrows indicate the positions of two lowerM r catabolites. C, Western blot probed with N terminus-specific antibody. Lanes are as in B. The arrow indicates the position of mature cytochrome f. D, Western blot probed with C terminus-specific antibody. Lanes are as in B. The arrow indicates the position of mature cytochrome f. All lanes were loaded with equal protein (5 μg).
- Fig. 6.
N-terminal microsequence (shown in boldface) of the larger, more abundant catabolite of cytochrome f in stromal lipid-protein particles from yellow wax bean (Pv; see Fig. 5) aligned with the amino acid sequence of mature cytochromef protein from broad bean (Vf). A gap (represented by a dash) has been introduced into the microsequence to align it with the sequence of the mature protein. The X in the microsequence represents an ambiguous residue. The numbers correspond to amino acid residues beginning with the mature N terminus. The sequences were aligned using MultAlin version 5.3.3 (Corpet, 1988), a multiple sequence alignment program available on the World Wide Web (www.toulouse.inra.fr).
- Fig. 7.
Western blot of a truncated recombinant form of cytochrome f, thylakoids, and stromal lipid-protein particles. The blot was probed with antibody raised against SDS-PAGE-purified mature, full-length cytochrome f. Lane 1, Full-length cytochrome f of thylakoids (5 μg of protein); lane 2, the larger more abundant cytochrome f catabolite of stromal lipid-protein particles (5 μg of protein); and lane 3, truncated recombinant cytochrome f (10 μg of protein).
