Levine T P, Rabouille C, Kieckbusch R H, Warren G
Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
J Biol Chem. 1996 Jul 19;271(29):17304-11. doi: 10.1074/jbc.271.29.17304.
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.
囊泡对接蛋白p115对间期高尔基体膜表现出可饱和的高亲和力结合。使用与有丝分裂细胞质预孵育的膜时，结合亲和力降低了20倍。相比之下，p115的有丝分裂预处理对结合没有影响。p115结合的减少是由磷酸化介导的，可由细胞周期蛋白依赖性激酶诱导，并且是完全可逆的。在对有丝分裂细胞进行分级分离后，还发现p115从膜转移到了细胞质中。通过体外有丝分裂孵育来探讨结合减少的功能意义，这种孵育会拆解高尔基体潴泡，主要产生运输囊泡。添加过量的p115可减少潴泡膜的损失，这表明在运输囊泡积累时，p115的作用是有限的。有丝分裂过程中高尔基体内运输的停止被认为是由于膜融合受到抑制，而运输囊泡的出芽仍在继续。这个过程也有助于有丝分裂高尔基体的拆解。我们的结果表明，高尔基体膜存在有丝分裂修饰，导致p115受体的亲和力降低。p115结合减少可能通过阻止先前的囊泡对接而在膜融合抑制中起作用。
