Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer.
The purified (H+)ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H(+)-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H+)ATPase molecules were closely associated with liposomes, exhibiting a high H(+)-pumping activity (150,000% quenching min-1.mg-1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H+)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H+)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles ("inside-out" orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H+)ATPase could also functionally insert into bilayer from PC:PE:PG or PC:PE:PI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H+)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H+)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation.
Simon-Plas
F
F
Biochimie et Physiologie Végétales, Institut National de la Recherche Agronomique, Ecole Nationale Supérieure Agronomique, Montpellier, France.
Venema
K
K
Grouzis
J P
JP
Gibrat
R
R
Rigaud
J
J
Grignon
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis / Q000494:pharmacology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D013025:Glycine max; D003313:Zea mays
D003902:Detergents; D008051:Lipid Bilayers; D008081:Liposomes; D010743:Phospholipids; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases