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Visualization by Freeze‐Fracture Electron Microscopy of Intramembraneous Particles corresponding to the Tonoplast H + ‐Pyrophosphatase and H + ‐ATPase of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie *
Author(s) -
Mariaux J.B.,
Becker Andrea,
Kemna Inge,
Ratajczak R.,
FischerSchliebs Elke,
Kramer D.,
Lüttge U.,
Marigo G.
Publication year - 1994
Publication title -
botanica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.871
H-Index - 87
eISSN - 1438-8677
pISSN - 0932-8629
DOI - 10.1111/j.1438-8677.1994.tb00802.x
Subject(s) - vesicle , vacuole , atpase , protein subunit , cytochemistry , electron microscope , tricine , chemistry , pyrophosphatase , crystallography , membrane , kalanchoe , biochemistry , biology , enzyme , botany , physics , cytoplasm , gene , optics
The H + ‐PPase and the H + ‐ATPase of the vacuolar membrane were separated during purification of tonoplast proteins of Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie. Three membrane protein fractions prepared contained firstly, the H + ‐PPase protein without any subunits of the H + ‐ATPase, secondly, the H + ‐PPase protein with only minute traces of the intramembraneous 16 kDa c‐subunit of the H + ‐ATPase, and thirdly, the H + ‐ATPase subunits without H + ‐PPase peptides as verified by SDS‐PAGE. These three preparations were reconstituted into soybean ( Glycine max L.)‐phospholipid vesicles, and compared with proteoliposomes obtained by reconstitution of total solubilized tonoplast proteins as well as with native tonoplast vesicles. Analysis of freeze‐fracture replicas prepared from these five different types of vesicles showed that there are two populations of intramembraneous particles, one with a diameter of 6.7‐7.2 nm corresponding to the H + ‐PPase, and one with an average diameter of 9.1 nm belonging to the H + ‐ATPase. Thus, freeze‐fracture electron microscopy allows one to visualize H + ‐PPase particles in addition to H + ‐ATPase particles in the tonoplast of Kalanchoë daigremontiana .