Visualization of antibody binding to the photosynthetic membrane: the transmembrane orientation of cytochrome b-559.
We have used immuno-gold labeling and electron microscopy to study the topography of thylakoid membrane polypeptides. Thylakoid vesicles formed by passage through a French press were adsorbed onto a plastic film supported by an electron microscope grid and processed for single or double immuno-gold labeling. After shadowing with platinum, the inside-out and right-side-out vesicles were identified by their distinctive morphologies. Right-side-out vesicles were labeled by a monoclonal antibody recognizing an epitope located in the trypsin-cleaved, N-terminal portion of the LHC II apoprotein, and by an antibody to CF1. A monoclonal antibody to the alpha-subunit of cytochrome b-559 reacted with a synthetic tridecapeptide corresponding to the C-terminal portion of the polypeptide. Both this antibody and a polyclonal antibody to the synthetic peptide labeled inside-out vesicles exclusively, indicating that the polypeptide C-terminus was exposed on the lumenal (exoplasmic) surface of the membrane.
Vallon
O
O
Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA.
Tae
G S
GS
Cramer
W A
WA
Simpson
D
D
Hoyer-Hansen
G
G
Bogorad
L
L
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis*; D002734:Chlorophyll / Q000378:metabolism*; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D003573:Cytochrome b Group / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007425:Intracellular Membranes / Q000648:ultrastructure; D045342:Light-Harvesting Protein Complexes; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins / Q000378:metabolism*; D010944:Plants / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003573:Cytochrome b Group; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D002734:Chlorophyll; C019313:cytochrome b559