Characterization of a cytosolic activity that induces the formation of Golgi membrane tubules in a cell-free reconstitution system.
Using a cell-free reconstitution system, we have characterized a cytosol- and ATP-dependent process that leads to the formation of membrane tubules from isolated Golgi complexes. These membrane tubules are uniform in diameter (50-70 nm) and morphologically identical to ones normally seen in cells and to those which are enhanced following brefeldin A treatment. Tubulation was strictly dependent on an activity present in an organelle-free extract of bovine brain cytosol and hydrolyzable ATP. Tubule formation was saturable with respect to both cytosol and ATP with half-maximal induction occurring at approximately 0.5 mg/mL cytosol and 10-20 microM ATP. Mild proteolytic treatment of Golgi membranes significantly reduced the extent of tubulation to subsequently added cytosol, suggesting that the tubulation activity interacts with Golgi-associated membrane proteins. The cytosolic tubulation activity was heat-labile, nondialyzable, and precipitated in ammonium sulfate. This activity could be followed through various chromatographic steps to yield fractions enriched in a major 40 kDa protein and several other minor proteins of approximately 80, 60, and 30 kDa. Monospecific antibodies against the 40K protein inhibited the cytosol-dependent tubulation of Golgi membranes in the cell-free system. Gel filtration chromatography suggests that the tubulation activity has a native molecular weight of approximately 125,000-140,000. These results establish the existence of cytosolic protein factors that regulate the formation of Golgi membrane tubules, and will provide the means for a biochemical dissection of membrane tubulation.
Banta
M
M
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Polizotto
R S
RS
Wood
S A
SA
de Figueiredo
P
P
Brown
W J
WJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure*; D002417:Cattle; D002458:Cell Fractionation; D002474:Cell-Free System; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003600:Cytosol / Q000502:physiology / Q000648:ultrastructure; D004734:Energy Metabolism; D006056:Golgi Apparatus / Q000648:ultrastructure*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008870:Microtubules / Q000648:ultrastructure*; D008970:Molecular Weight
D008565:Membrane Proteins; D000266:Adenylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate