Binding of the vesicle docking protein p115 to Golgi membranes is inhibited under mitotic conditions.
The vesicle docking protein p115 showed saturable, high affinity binding to interphase Golgi membranes. The affinity of binding was up to 20-fold lower using membranes preincubated with mitotic cytosol. In contrast, binding was not affected by mitotic pretreatment of p115. The reduction in p115 binding was mediated by phosphorylation, could be induced by a cyclin-dependent kinase, and was fully reversible. A shift of p115 from membranes to cytosol was also found after fractionating mitotic cells. The functional significance of the decreased binding was addressed by in vitro mitotic incubations which disassemble Golgi cisternae, predominantly producing transport vesicles. The addition of excess p115 decreased loss of membrane from cisternae, indicating that p115's action is limiting while transport vesicles accumulate. The cessation of intra-Golgi traffic in mitosis has been hypothesized to result from an inhibition of membrane fusion while budding of transport vesicles continues. This process also contributes to mitotic Golgi disassembly. Our results imply that there is a mitotic modification to Golgi membranes leading to a reduction in the affinity of the p115 receptor. Reduced p115 binding may play a part in the inhibition of membrane fusion by preventing prior vesicle docking.
Levine
T P
TP
Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Rabouille
C
C
Kieckbusch
R H
RH
Warren
G
G
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002453:Cell Cycle; D002458:Cell Fractionation; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D008938:Mitosis; D006656:Protamine Kinase / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D033921:Vesicular Transport Proteins
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C091580:vesicular transport factor p115; D006656:Protamine Kinase; D018844:Cyclin-Dependent Kinases