Biochemical demonstration of the involvement of fatty acyl-CoA synthetase in fatty acid translocation across the plasma membrane.
Fatty acyl-CoA synthetase, the first enzyme of the beta-oxidation pathway, has been proposed to be involved in long chain fatty acid translocation across the plasma membrane of prokaryotic and eukaryotic cells. To test this proposal, we used an in vitro system consisting of Escherichia coli inner (plasma) membrane vesicles containing differing amounts of trapped fatty acyl-CoA synthetase and its substrates CoA and ATP. This system allowed us to investigate the involvement of fatty acyl-CoA synthetase independently of other proteins that are involved in fatty acid translocation across the outer membrane and in downstream steps in beta-oxidation, because these proteins are not retained in the inner membrane vesicles. Fatty acid uptake in vesicles containing fatty acyl-CoA synthetase was dependent on the amount of exogenous ATP and CoASH trapped by freeze-thawing. The uptake of fatty acid in the presence of non-limiting amounts of ATP and CoASH was dependent on the amount of endogenous fatty acyl-CoA synthetase either retained within vesicles during isolation or trapped within vesicles after isolation by freeze-thawing. Moreover, the fatty acid taken up by the vesicles was converted to fatty acyl-CoA. These data are consistent with the proposal that fatty acyl-CoA synthetase facilitates long chain fatty acid permeation of the inner membrane by a vectorial thioesterification mechanism.
Schmelter
Tillmann
T
Department of Biochemistry, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada.
Trigatti
Bernardo L
BL
Gerber
Gerhard E
GE
Mangroo
Dev
D
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D019301:Oleic Acid / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D021381:Protein Transport; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D005227:Fatty Acids; D010743:Phospholipids; D019301:Oleic Acid; D019308:Palmitic Acid; D000255:Adenosine Triphosphate; D011392:Proline; D015094:3-Hydroxyacyl CoA Dehydrogenases; D001616:beta-Galactosidase; D003066:Coenzyme A Ligases; D010100:Oxygen; D003065:Coenzyme A