Vancurova I, Lou W, Paine T M, Paine P L
Department of Biological Sciences, St. John's University, Jamaica, NY 11439.
Eur J Cell Biol. 1993 Oct;62(1):22-33.
Specific proteins are selectively translocated into the cell nucleus and accumulated therein, but the molecular mechanisms underlying this fundamental eukaryotic transport process remain obscure. We have employed a new experimental system with notable advantages for resolving protein translocation and accumulation mechanisms. Individual nuclei are isolated from oocytes under mineral oil and conjoined under the oil with either an aqueous bead or a similar volume of oocyte cytoplasm to form closed transport pairs. Using these pairs one can (i) present transportant proteins via the bead or cytoplasm to a minimally disturbed nucleus, (ii) monitor the intactness of the nuclear envelope, and (iii) separate pairs at various times after formation and measure the amount of transportant in each compartment. In addition, it is uniquely possible with these pairs to determine whether or not a transportant's concentration gradient constitutes a chemical activity gradient. This is done by puncturing the envelope, thus eliminating its normal sieving restrictions on diffusion, and measuring the effect on the transportant distributions. We demonstrate that a prototypical nuclear-accumulating protein, nucleoplasmin (Np), is translocated through the nuclear pore complex by a mechanism of facilitated transport, rather than active transport. We further show that Np's high accumulation results from subsequent intranuclear binding. Np's facilitated transport and intranuclear binding are both ATP-dependent, and the latter requires cytoplasmic protein(s).
特定蛋白质会被选择性地转运到细胞核内并在其中积累，然而这种基本的真核生物转运过程背后的分子机制仍不清楚。我们采用了一种具有显著优势的新实验系统来解析蛋白质转运和积累机制。在矿物油下从卵母细胞中分离出单个细胞核，并在油中与水性珠子或等体积的卵母细胞细胞质相连，形成封闭的转运对。利用这些转运对，可以（i）通过珠子或细胞质将转运蛋白呈现给受干扰最小的细胞核，（ii）监测核膜的完整性，以及（iii）在形成后的不同时间分离转运对，并测量每个隔室中转运蛋白的量。此外，利用这些转运对还能独特地确定转运蛋白的浓度梯度是否构成化学活性梯度。这通过刺穿核膜来实现，从而消除其对扩散的正常筛分限制，并测量对转运蛋白分布的影响。我们证明，一种典型的核积累蛋白，核质素（Np），是通过易化转运机制而非主动转运穿过核孔复合体的。我们进一步表明，Np的高积累是由于随后的核内结合。Np的易化转运和核内结合都依赖于ATP，并且后者需要细胞质蛋白。
